SureSelect Cancer CGP Assay

User Guide

For Research Use Only. Not for use in diagnostic procedures.

Version D0, April 2024

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SureSelect Cancer CGP Assay User Guide

 In This Guide…
This guide provides instructions for the SureSelect Cancer CGP Assay, a targeted next-generation
sequencing (NGS) solution for interrogation of genomic and transcriptomic features of relevance
in solid tumors. Support is also provided for SureSelect Cancer Tumor-Specific and Custom Panel
Assays, performed using minor protocol modifications.

1 Before You Begin

2 RNA-Specific Workflow Steps

3 DNA-Specific Workflow Steps

4 DNA/RNA Workflow Steps: Library Prep and Hybridization to SureSelect Cancer CGP Assay
Probes

5 NGS and Analysis Workflow Steps

6 Appendix 1: SureSelect Cancer CGP Automation

7 Appendix 2: SureSelect Cancer Tumor-Specific Assays

8 Appendix 3: SureSelect Cancer Custom Panel Assays

9 Reference

SureSelect Cancer CGP Assay User Guide 3

 What’s New in Version D0
• Support for SureSelect Cancer Custom Assays (see page 8 and “Appendix 3: SureSelect
Cancer Custom Panel Assays" on page 70)
• Access information for the SureSelect XT HS2 Index Sequence Resource Excel spreadsheet
(see page 79)

What’s New in Version C0

• Support for Agilent’s Alissa Reporter analysis software v1.3 (see page 8, page 9, page 10
page 11, page 48, and page 51 to page 58).
• Update to Cancer CGP DNA Assay MSI target and coverage descriptions (see page 8 and
page 58)
• Updates to Magnis automation RNA assay and Magnis firmware v1.4 availability information
(see page 59 and page 60)
• Updates to Bravo automation information for NGS Bravo Option B+ users (see footnote to
Table 47 on page 61)

What’s New in Version B0

• Support for new SureSelect Cancer Tumor-Specific Assays (see page 8 and “Appendix 2:
SureSelect Cancer Tumor-Specific Assays" on page 67)
• Update to Cancer CGP Assay CNV target description to 32 loci (see page 8)
• Correction of typographical errors in Note on page 25
• Updates to downstream sequencing support information (see page 51 to page 52)
• Updates to Notices section

SureSelect Cancer CGP Assay User Guide 4

Content

1 Before You Begin 8

Introduction to the SureSelect Cancer CGP Assay 8
Sample requirements 9
Overview of the Workflow 10
SureSelect Cancer CGP Assay Components 11
Additional Materials Required 12
Procedural and Safety Notes 14

2 RNA-Specific Workflow Steps 16

Step 1. Prepare and qualify RNA samples 17
FFPE RNA samples 17
Intact RNA samples 18
Step 2. Add 2X Priming Buffer to all samples and fragment intact RNA samples 19
Step 3. Synthesize first-strand cDNA 19
Step 4. Synthesize second-strand cDNA 20
Step 5. Purify cDNA using AMPure XP Beads 21

3 DNA-Specific Workflow Steps 23

Step 1. Prepare and qualify the genomic DNA samples 24
FFPE DNA samples 24
Intact DNA samples 25
Step 2. Fragment the DNA 25
Method 1: Enzymatic DNA fragmentation 25
Method 2: Mechanical DNA shearing with Covaris 27

4 DNA/RNA Workflow Steps: Library Prep and Hybridization to SureSelect Cancer CGP
Assay Probes 29
Library Preparation and Pre-capture Amplification 30
Step 1. Prepare the ligation master mix 31
Step 2. Repair and dA-tail the DNA 3' ends 31
Step 3. Ligate the molecular-barcoded adaptor 32
Step 4. Purify libraries using AMPure XP Beads 33
Step 5. Amplify the pre-capture libraries 34
Step 6. Purify amplified libraries using AMPure XP Beads 36
Step 7. QC and quantify the pre-capture libraries 37

SureSelect Cancer CGP Assay User Guide 5

 Hybridization, Capture and Post-capture Amplification 39
Step 1. Hybridize libraries to the SureSelect Cancer CGP Assay Probe 40
Step 2. Prepare streptavidin beads for capture 42
Step 3. Capture the hybridized libraries 42
Step 4. Amplify the captured libraries 43
Step 5. Purify the final libraries using AMPure XP Beads 44
Step 6. QC and quantify final libraries 46

5 NGS and Analysis Workflow Steps 48

Step 1. Pool samples for multiplexed sequencing 49
Step 2. Prepare the sequencing samples 50
Step 3. Sequence the libraries 51
Step 4. Process the reads to analysis-ready files 52
Step 5. Analyze using Alissa Reporter software 53
Analysis Considerations 57

6 Appendix 1: SureSelect Cancer CGP Automation 59

Automation Overview 59
Magnis Automation Workflow 60
Pre-run instrument and labware preparation 60
Pre-run sample preparation 60
Performing the Magnis NGS library preparation run 61
Post-run library processing 61
Bravo Automation Workflow 61
Pre-run sample preparation and qualification 62
Performing the NGS library preparation run 62
RNA Assay automation protocols 63
DNA Assay automation protocols 65
Post-run library processing 66

7 Appendix 2: SureSelect Cancer Tumor-Specific Assays 67

Overview of SureSelect Cancer Tumor-Specific Assays 67
Materials Required for the SureSelect Cancer Assay 67
Running the SureSelect Cancer Assay 69

8 Appendix 3: SureSelect Cancer Custom Panel Assays 70

Overview of SureSelect Cancer Custom Panel Assays 70
Materials Required for SureSelect Cancer Custom Panel Assays 70
Running a SureSelect Cancer Custom Panel Assay 72

SureSelect Cancer CGP Assay User Guide 6

 9 Reference 74
Reagent Kit Contents 75
SureSelect XT HS2 Index Primer Pair Information 79
SureSelect XT HS2 Index Primer Pair Sequences 80
Index Primer Pair Strip Tube and Plate Maps 84
Troubleshooting Guide 86

SureSelect Cancer CGP Assay User Guide 7

 Agilent SureSelect Cancer CGP Assay
User Guide

1
Before You Begin
Introduction to the SureSelect Cancer CGP Assay 8
Overview of the Workflow 10
SureSelect Cancer CGP Assay Components 11
Additional Materials Required 12
Procedural and Safety Notes 14

Introduction to the SureSelect Cancer CGP Assay

The SureSelect Cancer Comprehensive Genomic Profiling (CGP) Assay is a targeted

next-generation sequencing (NGS) solution that enables interrogation of genomic and
transcriptomic regions of relevance in solid tumors for a variety of features including those listed
below.
• DNA SNVs (single nucleotide variations) and Indels (short insertions and deletions): The
DNA Assay probe design includes full exonic coverage of 679 cancer-associated genes,
allowing comprehensive cancer gene variant calling against the reference genome.
• DNA CNVs (copy number variations): The DNA Assay probe design includes optimal target
sequence placement at 32 loci for detection of DNA amplification and deletion at key loci.
• DNA Translocations: The DNA Assay probe design includes intronic coverage in 12 genes
to enable translocation detection at key loci.
• Tumor Mutational Burden (TMB): Coverage of >1.6 Mb of coding sequence enables
quantification of TMB in DNA samples.
• Microsatellite Instability (MSI): The DNA assay probe targets 288 sites available for
microsatellite instability (MSI) determinations, with coverage for typical samples in the
range of 250-280 sites, enabling quantification of MSI in DNA samples.
• RNA fusions and exon-skipping RNA splice variants: The RNA Assay probe design enables
detection of RNA fusions in 80 key genes (regardless of partner) and of important splice
variants EGFRvIII and MET Exon14-skipping.
The assay includes SureSelect XT HS2 reagents for preparation of target-enriched NGS libraries
from gDNA and total RNA samples. The workflow is summarized in Figure 1.
Once sequencing data is collected for the assay samples, Agilent offers Alissa Reporter analysis
applications tailored to the SureSelect Cancer Comprehensive Genomic Profiling (CGP) Assays.
See page 53 for more information. Alternative NGS analysis software tools can also be used for
variant discovery.
The protocols provided in this publication may also be used to interrogate gDNA samples using
Agilent’s SureSelect Cancer Tumor-Specific Assays (see page 67) or SureSelect Cancer Custom
Panel Assays (see page 70).

8
 Sample requirements
The SureSelect Cancer CGP Assay supports analysis of DNA and RNA samples isolated from
fresh or fresh-frozen samples or extracted from formalin-fixed, paraffin-embedded (FFPE)
tissues. The assay is optimized for sample input amounts of 50 ng genomic DNA or 50 ng total
RNA. For low-quality FFPE samples, assay performance may be improved by increasing the
amount of DNA or RNA input to up to 200 ng. Use of 10–200 ng DNA or RNA input is supported by
the SureSelect XT HS2 system; however, use of input <50 ng for the SureSelect Cancer CGP
Assay may lead to lower target coverage and reduced detection of low-frequency variants. See
Troubleshooting on page 86 for more information on use of low input (<50 ng) samples.
FFPE samples should be isolated from a minimum of 3 tissue block sections of 5 µm each and
containing ≥15% tumor content. Agilent has not validated the SureSelect Cancer CGP Assay using
liquid biopsy or needle aspiration samples. See Troubleshooting on page 88 for more information
on use of unsupported sample types.
Consult the selected analysis software guidelines for any additional sample requirements. The
assay supports tumor-normal paired analysis using matched or unmatched reference DNA where
variant analysis may require specific types or numbers of reference samples. For example, CNV
analysis algorithms typically require co-processing of matched or unmatched reference DNA
samples without copy number aberrations in the regions of interest. Reference DNA samples may
be isolated from normal FFPE tissue blocks, fresh-frozen tissues or cell lines. Agilent’s OneSeq
Human Reference DNA is recommended for use as an unmatched reference DNA sample.

Sample requirements for Agilent’s Alissa Reporter Cancer CGP applications

For analysis using Alissa Reporter, some types of variant analysis require co-processing of the
tumor (Target) sample and a matched/unmatched normal (Reference) sample.
SNV/Indel analysis in Alissa Reporter can be completed in Tumor-normal or Tumor-only mode.
Use of a matched/unmatched normal Reference sample is required for analysis in Tumor-normal
mode, where the Reference sample is used to help filter germline variants. Using a matched
reference sample for Tumor-normal mode analysis is recommended since detection of somatic
variants can be less sensitive when an unmatched reference is used. When no Reference sample
is available, analysis is performed in Tumor-only mode with reduced germline variant filtration.
CNV analysis in Alissa Reporter always requires co-processing of a matched/unmatched
Reference sample with each tumor Target sample.
For best results, process the Target and Reference samples to be co-analyzed in Alissa Reporter
in the same SureSelect run and in the same sequencing run in order to minimize any
processing-based variance. The software allows analysis using pre-established unmatched
reference sample data from a prior run but potential bias due to batch differences may negatively
impact the accuracy of calling.
For variant calling on the X and Y chromosomes, it is important to use Target and Reference
samples of the same sex.

SureSelect Cancer CGP Assay User Guide 9

Overview of the Workflow

SureSelect Cancer SureSelect Cancer

CGP Assay for DNA CGP Assay for RNA
Fragment RNA (0.5 hr)
(intact samples only)

Prepare 1st strand cDNA synthesis (1 hr)

Fragment
gDNA or
genomic DNA
cDNA (1 hr)
Fragments 2nd strand cDNA synthesis (1.5 hr)

cDNA cleanup (0.5 hr)

Day 1

End repair / Adaptor ligation (1.5 hr)

Library cleanup (0.5 hr)

Library
Pre-capture PCR (1 hr)
Preparation

Library cleanup (0.5 hr)

Library QC (time varies)

Hybridization and capture (3.5 hrs)

Post-capture PCR (1 hr)

Day 2

Target
Enrichment
Library cleanup (0.5 hr)

Library QC (time varies)

Day 3

Sample pooling
7IUYIRGMRK
%REP]WMW
SequencingERHEREP]WMWYWMRK%PMWWE6ITSVXIVSVEPXIVREXMZIXSSP

Optional stopping point

Figure 1 SureSelect Cancer CGP assay workflow. DNA and RNA samples are processed in sep-
arate reactions throughout the NGS library preparation and target enrichment steps, but can be
processed in parallel beginning with the end repair/adaptor ligation workflow segment and can
be sequenced and analyzed together. See page 16 for synchronization guidelines. The provid-
ed time estimates are for processing up to 16 reactions per run; your results may vary.

SureSelect Cancer CGP Assay User Guide 10

SureSelect Cancer CGP Assay Components

The SureSelect Cancer CGP Assay requires the components listed below:
• SureSelect Cancer CGP Assay Probes (DNA and RNA assay probes in separate vials)
• Library preparation and hybridization/capture reagents using SureSelect XT HS2 chemistry
• Optional: Alissa Reporter analysis software (Agilent p/n G5393AA for use of Alissa Cancer CGP
DNA assay, p/n G5394AA for use of Alissa Cancer CGP RNA assay, or Agilent p/n M5711AA for
use of both DNA and RNA assays). Please email informatics_support@agilent.com or contact
your local representative for software access information.
Table 1 shows the SureSelect Cancer CGP Assay Kit formats available for non-automated sample
processing. Kits for automated processing are described in Table 44 on page 59.
See Table 2 through Table 4 for additional materials required to complete the assay protocols.

Table 1 Ordering information for SureSelect Cancer CGP Assay components

Description Agilent Reagent Modules Included*

Part
Probe(s) DNA Library RNA Library Capture Beads, Enzymatic DNA Reference DNA/
Number
Prep + Hyb Prep + Hyb Purification Fragmentation Control RNA
Reagents Reagents Beads

Complete Starter Kit for DNA & RNA Assays (16 Samples for each assay)
SureSelect Cancer CGP G9965A DNA     
Assay Starter Kit DNA & RNA, & RNA† (Index 1-16) (Index 17-32)
16 Samples Each (32 Hyb)

DNA & RNA Assay Kit (96 Samples for each assay)
SureSelect Cancer CGP G9966A DNA    — —
Assay DNA & RNA Kit, & RNA† (Index 1-96) (Index 97-192) (see Table 3 (see Table 3
96 Samples Each (192 Hyb) on page 13) on page 13)

DNA Assay Kits (16 Samples or 96 Samples)

SureSelect Cancer CGP G9967A DNA†  ×  — —
Assay DNA Kit, 16 Samples (Index 1-16) (not required) (see Table 3 (see Table 3
on page 13) on page 13)

SureSelect Cancer CGP G9967B DNA†  ×  — —

Assay DNA Kit, 96 Samples (Index 1-96) (not required) (see Table 3 (see Table 3
on page 13) on page 13)

RNA Assay Kits (16 Samples or 96 Samples)

SureSelect Cancer CGP G9968A RNA† ×   × —
Assay RNA Kit, 16 Samples (not required) (not required) (see Table 3
(Index 17-32)
on page 13)

SureSelect Cancer CGP G9968B RNA† ×   × —

Assay RNA Kit, 96 Samples (not required) (Index 97-192) (not required) (see Table 3
on page 13)

* See “Reagent Kit Contents” on page 75 through page 78 for a complete list of Reagent Kit components provided with each product.

† The SureSelect Cancer CGP Assay Probes may also be purchased separately. See Table 59 on page 77 for part number information.

SureSelect Cancer CGP Assay User Guide 11

Additional Materials Required

Use the tables below to select the additional materials required to complete the SureSelect
Cancer CGP Assay. Table 2 lists the materials needed for all workflows, while Table 3 and Table 4
list additional materials needed for specific sample types and protocol step options.

Sample volumes exceed 0.2 mL in certain steps of this protocol. Make sure that the plasticware used with
CAU TIO N the selected thermal cycler holds 0.25 mL per well.

Table 2 Required Equipment and Reagents--All Sample Types/Fragmentation Methods

Description Vendor and Part Number

Thermal Cycler with 96-well, 0.2 mL block Various suppliers
Plasticware compatible with the selected thermal cycler: Consult the thermal cycler manufacturer’s recommendations
96-well plates or 8-well strip tubes
Tube cap strips
Nucleic acid analysis system (instrument and consumables) Select one system from Table 4 on page 14
Small-volume spectrophotometer NanoDrop 2000, Thermo Fisher Scientific p/n ND-2000 or
equivalent
Qubit Fluorometer Thermo Fisher Scientific p/n Q33238
Qubit Assay Tubes Thermo Fisher Scientific p/n Q32856
Qubit BR dsDNA Assay Kit Thermo Fisher Scientific
100 assays p/n Q32850
500 assays p/n Q32853
Nuclease-free Water (not DEPC-treated) Thermo Fisher Scientific p/n AM9930
1X Low TE Buffer (10 mM Tris-HCl, pH 7.5-8.0, 0.1 mM EDTA) Thermo Fisher Scientific p/n 12090-015, or equivalent
100% Ethanol (Ethyl Alcohol, 200 proof) Millipore p/n EX0276
DNA LoBind Tubes, 1.5-mL PCR clean, 250 pieces Eppendorf p/n 022431021 or equivalent
Microcentrifuge Eppendorf microcentrifuge, model 5417C or equivalent
Plate or strip tube centrifuge Labnet International MPS1000 Mini Plate Spinner, p/n C1000
(requires adapter, p/n C1000-ADAPT, for use with strip tubes)
or equivalent
96-well plate mixer Eppendorf ThermoMixer C, p/n 5382000023 and Eppendorf
SmartBlock 96 PCR, p/n 5306000006, or equivalent
Magnetic separator Thermo Fisher Scientific p/n 12331D or equivalent*
Multichannel and single channel pipettes Rainin Pipet-Lite Multi Pipette or equivalent
Sterile, nuclease-free aerosol barrier pipette tips, vortex mixer, General laboratory supplier
ice bucket, and powder-free gloves

* Select a magnetic separator configured to collect magnetic particles on one side of each well. Do not use a magnetic separator configured to
collect the particles in a ring formation.

SureSelect Cancer CGP Assay User Guide 12

Table 3 Additional Required Materials based on Sample Type/Fragmentation Method

Description Vendor and Part Number Usage Notes

Required for DNA assays (not required for RNA-only assays)
FFPE gDNA purification system, for example: QIAGEN Recommended system for FFPE gDNA
QIAamp DNA FFPE Tissue Kit, 50 Samples p/n 56404 sample purification.
Deparaffinization Solution p/n 19093
FFPE DNA integrity assessment system: Recommended systems for FFPE
Agilent NGS FFPE QC Kit Agilent gDNA qualification prior to library
16 reactions p/n G9700A preparation.
96 reactions p/n G9700B
OR
TapeStation Genomic DNA Analysis Consumables: Agilent
Genomic DNA ScreenTape p/n 5067-5365
Genomic DNA Reagents p/n 5067-5366
High-quality gDNA purification system, for example: Recommended system for purification
QIAamp DNA Mini Kit QIAGEN of intact gDNA.
50 Samples p/n 51304
250 Samples p/n 51306
OneSeq Human Reference DNA, Female Agilent p/n 5190-8850 Control and unmatched reference DNA
SureSelect Enzymatic Fragmentation Kit Agilent Not required for workflows using
p/n 5191-4080 (96 reactions) mechanical (Covaris-mediated) DNA
shearing.
Mechanical DNA fragmentation system: Not required for workflows using
Covaris Sample Preparation System Covaris model E220 enzymatic DNA fragmentation.
Covaris microTUBE sample holders Covaris p/n 520045 Additional Covaris instrument models
and sample holders may be used after
optimization of shearing conditions.
Required for RNA assays (not required for DNA-only assays)
FFPE RNA purification system, for example: QIAGEN Recommended system for FFPE RNA
RNeasy FFPE Kit, 50 Samples p/n 73504 sample purification.
FFPE RNA integrity analysis system: SeeTable 4 on page 14 for Select the RNA analysis consumables
4200/4150 TapeStation with RNA ordering information designed for the qualification system
ScreenTape/High-Sensitivity RNA ScreenTape used in your laboratory and appropriate
OR for the RNA concentrations of your
2100 Bioanalyzer with RNA 6000 Pico/Nano Kit samples.
OR
5200/5300/5400 Fragment Analyzer with RNA/HS
RNA Kit
QPCR Human Reference Total RNA Agilent p/n 750500 Control RNA

SureSelect Cancer CGP Assay User Guide 13

Table 4 Nucleic Acid Analysis Platform Options--Select One

Analysis System Vendor and Part Number Information

Agilent 4200/4150 TapeStation Instrument Agilent p/n G2991AA/G2992AA
Consumables:
96-well sample plates p/n 5042-8502
96-well plate foil seals p/n 5067-5154
8-well tube strips p/n 401428
8-well tube strip caps p/n 401425
D1000 ScreenTape p/n 5067-5582
D1000 Reagents p/n 5067-5583
High Sensitivity D1000 ScreenTape p/n 5067-5584
High Sensitivity D1000 Reagents p/n 5067-5585
RNA ScreenTape p/n 5067-5576
RNA ScreenTape Sample Buffer p/n 5067-5577
RNA ScreenTape Ladder p/n 5067-5578
High Sensitivity RNA ScreenTape p/n 5067-5579
High Sensitivity RNA ScreenTape Sample Buffer p/n 5067-5580
High Sensitivity RNA ScreenTape Ladder p/n 5067-5581
Agilent 2100 Bioanalyzer Instrument p/n G2939BA
Agilent 2100 Expert SW Laptop Bundle (optional) p/n G2953CA
Consumables:
DNA 1000 Kit p/n 5067-1504
High Sensitivity DNA Kit p/n 5067-4626
RNA 6000 Pico Kit p/n 5067-1513
RNA 6000 Nano Kit p/n 5067-1511
Agilent 5200/5300/5400 Fragment Analyzer Instrument Agilent p/n M5310AA/M5311AA/M5312AA
Consumables:
NGS Fragment Kit (1-6000 bp) p/n DNF-473-0500
HS NGS Fragment Kit (1-6000 bp) p/n DNF-474-0500
RNA Kit (15NT) p/n DNF-473-0500
HS RNA Kit (15NT) p/n DNF-474-0500

Procedural and Safety Notes

• Use best-practices to prevent PCR product and nuclease contamination of samples

throughout the workflow:
1 Assign separate pre-PCR and post-PCR work areas and use dedicated equipment, supplies,
and reagents in each area. In particular, never use materials designated to post-PCR work
areas for pre-PCR segments of the workflow.
2 Maintain clean work areas. Clean the surfaces that pose the highest risk of contamination
daily using a 10% bleach solution, or equivalent.
3 Always use dedicated pre-PCR pipettors with nuclease-free aerosol-resistant tips to pipette
dedicated pre-PCR solutions.
4 Wear powder-free gloves. Use good laboratory hygiene, including changing gloves after
contact with any potentially-contaminated surfaces.
• Several reagent solutions used in the SureSelect Cancer CGP Assay protocols are highly
viscous. Make sure to follow the mixing instructions provided in the protocols.

SureSelect Cancer CGP Assay User Guide 14

 • For each protocol step that requires removal of tube cap strips, reseal the tubes with a fresh
strip of caps. Cap deformation may result from exposure of the cap strips to the heated lid of
the thermal cycler and from other procedural steps. Reuse of strip caps can cause sample
loss, sample contamination, or imprecision in sample temperatures during thermal cycler
incubation steps.
• Possible stopping points, where samples may be stored at 4°C or –20°C, are marked in the
protocol. Do not subject the samples to multiple freeze/thaw cycles.
• In general, follow Biosafety Level 1 (BSL1) safety rules.

Wear appropriate personal protective equipment (PPE) when working in the

CAU TIO N laboratory.

SureSelect Cancer CGP Assay User Guide 15

Agilent SureSelect Cancer CGP Assay
User Guide

2
RNA-Specific Workflow Steps
Step 1. Prepare and qualify RNA samples 17
Step 2. Add 2X Priming Buffer to all samples and fragment intact RNA samples 19
Step 3. Synthesize first-strand cDNA 19
Step 4. Synthesize second-strand cDNA 20
Step 5. Purify cDNA using AMPure XP Beads 21

This section describes the steps to prepare fragmented input RNA and the steps to convert the
RNA fragments to strand-specific cDNA prior to sequencing library preparation. The protocols
include conditions for FFPE-derived RNA samples (see page 17) and intact RNA from fresh or
fresh-frozen samples (see page 18).
FFPE-derived RNA samples are already sufficiently fragmented for library preparation, while the
intact RNA samples are chemically-fragmented in this step. The protocol produces cDNA
fragments for the SureSelect Cancer CGP RNA Assay suitable for 2 x 150 read length NGS.

For FFPE RNA samples, initial RNA fragment size may impact the size distribution in the
N OTE final cDNA library, with some library fragments shorter than 150 bp.

Guidelines for simultaneous DNA & RNA workflows

If you are preparing DNA libraries (only), proceed directly to page 23.
If you are preparing both DNA and RNA libraries in the same run, review both the RNA-specific
steps in this section and the DNA-specific steps on page 23 through page 28 before you begin.
Once both input gDNA and total RNA samples have been prepared and qualified, the DNA & RNA
assay workflows can be synchronized by starting with the RNA-specific workflow steps (page 17
to page 21) where RNA samples are processed to purified cDNA and stored as directed on
page 22 while DNA samples are fragmented.

16
 This workflow segment uses the components listed in Table 5. Remove the listed reagents from
cold storage, and prepare as directed before use (refer to the Where Used column).

Table 5 Reagents thawed before use in protocol

Storage Location Kit Component Preparative Steps Where Used

2X Priming Buffer (tube with purple cap) Thaw on ice then keep on ice, vortex to mix page 19

First Strand Master Mix (amber tube with Thaw on ice for 30 minutes then keep on page 19
SureSelect cDNA amber cap)* ice, vortex to mix
Module (Pre PCR), Second Strand Enzyme Mix (tube with blue Thaw on ice then keep on ice, vortex to mix page 20
–20°C cap or bottle)

Second Strand Oligo Mix (tube with yellow Thaw on ice then keep on ice, vortex to mix page 20
cap)

+4°C SureSelect RNA AMPure XP Beads Equilibrate at room temperature for at page 21
least 30 minutes before use, vortex to mix

* The First Strand Master Mix contains actinomycin D and is provided ready-to-use. Keep the reagent in the supplied amber vial to protect the
contents from exposure to light.

Step 1. Prepare and qualify RNA samples

FFPE RNA samples

The instructions in this section are for FFPE-derived RNA samples. For intact (non-FFPE) RNA
samples, instead follow the instructions on page 18.
Samples are obtained from tissue resection (tissue curls or sections on slide), with use of a
minimum of 3 sections of 5 µm each and with ≥15% tumor content (measured by haemotoxylin &
eosin staining) recommended.
1 Prepare total RNA from each FFPE sample in the run. The optimized library preparation
protocol uses 50 ng of FFPE total RNA in a 10 µL volume of nuclease-free water. The assay
may be performed using up to 200 ng input RNA.
The SureSelect XT HS2 RNA system supports use of 10–200 ng RNA input. Use of
N OTE <50 ng RNA for the SureSelect Cancer CGP Assay may reduce yield and target coverage.

2 Use a small-volume spectrophotometer to determine the RNA concentration and the 260/280
and 260/230 absorbance ratio values for the sample. High-quality RNA samples are indicated
by values of approximately 1.8 to 2.0 for both ratios.
3 Examine the starting RNA size distribution in the sample using one of the RNA qualification
systems described in Table 6. Select the specific assay appropriate for your sample based on
the RNA concentration determined in step 2.
Table 6 Agilent RNA qualification platforms

Analysis Instrument RNA Qualification Assay Analysis Mode

4200/4150 TapeStation RNA Screen Tape or High Sensitivity RNA Region analysis using TapeStation Analysis Software
Screen Tape
2100 Bioanalyzer RNA 6000 PicoChip or NanoChip Smear/Region analysis using 2100 Expert Software
5200 Fragment Analyzer RNA Kit (15NT) or HS RNA Kit (15NT) Analysis using ProSize Data Analysis Software

SureSelect Cancer CGP Assay User Guide 17

 Grading of FFPE RNA quality by RNA Integrity Number (RIN) is not recommended
N OTE for this application.

Determine the DV200 (percentage of RNA in the sample that is >200 nt) using the analysis
mode described in Table 6. RNA molecules must be >200 nt for efficient conversion to cDNA
library. Consult Table 7 for DV200-based RNA input recommendations.
Table 7 RNA input guidelines based on DV200 score

DV200 Score RNA Input Guidelines

DV200 20% 50 ng RNA recommended (up to 200 ng may be used)
DV200 < 20% Not recommended for further processing

4 Place 50 ng of each FFPE RNA sample in 10 µL of nuclease-free water into wells of a thermal
cycler-compatible strip tube or PCR plate and hold on ice.
FFPE RNA library preparation steps continue in “Step 2. Add 2X Priming Buffer to all samples
and fragment intact RNA samples” below.

Intact RNA samples

The instructions in this section are for intact (non-FFPE) RNA samples. For FFPE-derived RNA
samples, see page 17.
1 Prepare intact total RNA from each fresh or fresh-frozen sample. The optimized library
preparation protocol uses 50 ng of total RNA in a 10 µL volume of nuclease-free water.
The SureSelect XT HS2 RNA system supports use of 10–200 ng RNA input. Use of
N OTE <50 ng RNA for the SureSelect Cancer CGP Assay may reduce yield and target coverage.

2 Use a small-volume spectrophotometer to determine the RNA concentration and the 260/280
and 260/230 absorbance ratio values for the sample. High-quality RNA samples are indicated
by values of approximately 1.8 to 2.0 for both ratios.
3 Place 50 ng of each intact RNA sample in 10 µL of nuclease-free water into wells of a thermal
cycler-compatible strip tube or PCR plate and hold on ice.
Studies investigating FFPE-derived experimental samples should also include a well
characterized, intact control RNA sample, in order to differentiate performance issues related to
sample quality from other factors. Agilent’s QPCR Human Reference Total RNA (supplied at
1 µg/µL) is recommended for this purpose. Dilute to 5 ng/µL in nuclease-free water before use.
Intact RNA samples and FFPE RNA samples must be placed in a separate strip tubes or
N OTE PCR plates, since these sample types are processed under different conditions in the
following section. After fragmentation of intact RNA, samples can be reformatted for
co-processing on a single plate or strip, beginning with first-strand cDNA synthesis on
page 19.

SureSelect Cancer CGP Assay User Guide 18

Step 2. Add 2X Priming Buffer to all samples and fragment intact
RNA samples

In this step, all RNA samples (both FFPE-derived and intact) are combined with 2X Priming Buffer,
containing primers used for cDNA synthesis in addition to fragmentation agents. The intact RNA
samples, only, are then chemically-fragmented by incubation at elevated temperature. The
FFPE-derived RNA samples are already sufficiently fragmented for library preparation and are
held on ice after 2X Priming Buffer addition to prevent further fragmentation.
1 Add 10 µL of 2X Priming Buffer to each RNA sample well, containing 50 ng of either FFPE RNA
or intact RNA. Mix well then spin briefly and hold the samples on ice.
2 Transfer the intact RNA samples to a thermal cycler and run the program in Table 8.
Leave the FFPE-derived RNA samples on ice during this step.
Table 8 Thermal cycler program for fragmentation of intact RNA samples (20 l vol)

Step Temperature Time

Step 1 94°C 4 minutes
Step 2 4°C 1 minute
Step 3 4°C Hold

3 Once the thermal cycler program reaches the 4°C Hold step, transfer the fragmented RNA
samples to ice.
Proceed immediately to “Step 3. Synthesize first-strand cDNA” to continue processing all RNA
samples.

Step 3. Synthesize first-strand cDNA

The First Strand Master Mix used in this step is viscous. Mix thoroughly by vortexing where
CAU TIO N directed in the protocol. Pipetting up and down is not sufficient to mix this reagent.
The First Strand Master Mix is provided with actinomycin D already supplied in the mixture.
Do not supplement with additional actinomycin D.

1 Preprogram a thermal cycler as shown in Table 9; pause until use in step 5.

Table 9 Thermal cycler program for first-strand cDNA synthesis (28 l vol)

Step Temperature Time

Step 1 25°C 10 minutes

Step 2 37°C 40 minutes

Step 3 4°C Hold

2 Vortex the thawed vial of First Strand Master Mix for 5 seconds at high speed to ensure
homogeneity.

SureSelect Cancer CGP Assay User Guide 19

 3 Add 8.5 µL of First Strand Master Mix to each RNA sample well.
4 Mix well by pipetting up and down 15–20 times or seal the wells and vortex at high speed for
5–10 seconds. Spin briefly to collect the liquid.
5 Place the samples in the thermal cycler, and resume the program in Table 9.

Step 4. Synthesize second-strand cDNA

The Second Strand Enzyme Mix used in this step is viscous. Mix thoroughly by vortexing
CAU TIO N where directed in the protocol. Pipetting up and down is not sufficient to mix this reagent.

1 Once the thermal cycler program in Table 9 begins the 4°C hold step, transfer the samples to
ice.
2 Preprogram a thermal cycler as shown in Table 10; pause until use in step 7.
Table 10 Thermal cycler program for second-strand synthesis (58 l vol)

Step Temperature Time

Step 1 16°C 60 minutes

Step 2 4°C Hold

3 Vortex the thawed vials of Second Strand Enzyme Mix and of Second Strand Oligo Mix at high
speed for 5 seconds to ensure homogeneity.
4 Add 25 µL of Second Strand Enzyme Mix to each sample well. Keep on ice.
5 Add 5 µL of Second Strand Oligo Mix to each sample well, for a total reaction volume of
58.5 µL. Keep on ice.
6 Mix well by pipetting up and down 15–20 times or seal the wells and vortex at high speed for
5–10 seconds. Spin briefly to collect the liquid.
7 Place the plate or strip tubes in the thermal cycler, and resume the program in Table 10.
The AMPure XP Beads used in the next step must be equilibrated to room temperature for
N OTE at least 30 minutes before use.

SureSelect Cancer CGP Assay User Guide 20

Step 5. Purify cDNA using AMPure XP Beads

Once the thermal cycler program in Table 10 reaches the 4°C hold step, purify the cDNA using
room temperature AmpPure XP Beads.
Critical purification protocol parameters are summarized for experienced users in Table 11. A
video demonstrating the AmpPure XP Bead purification protocol is available at Agilent.com.
(Perform all purification steps in plates or strip tubes as described below; do not transfer samples
to 1.5 ml tubes as shown in the video demonstration.)

Table 11 AMPure XP bead cDNA cleanup parameters

Parameter Value
Volume of RT AMPure XP bead suspension added to each sample well 105 µL
Final elution solvent and volume 52 µL nuclease-free water
Amount of eluted sample transferred to fresh well Approximately 50 µL

1 Prepare 400 µL of 70% ethanol per sample, plus excess, for use in step 8.
The freshly-prepared 70% ethanol may be used for all purification steps run on the same
N OTE day. Consult the workflow summary on page 10 to determine how many same-day
purification steps will be run.

2 Mix the room-temperature AMPure XP Beads well until homogeneous and consistent in color.
3 Transfer the cDNA samples from the thermal cycler to room temperature, then add 105 µL of
the bead suspension to each sample well.
4 Mix by pipetting up and down 15–20 times or cap the wells and vortex at high speed for
5–10 seconds then spin briefly to collect the samples, being careful not to pellet the beads.
5 Incubate the bead suspensions for 5 minutes at room temperature.
6 Put the plate or strip tube into a magnetic separation device. Wait for the solution to clear
(approximately 2 to 5 minutes).
7 Keep the plate or strip tube in the magnetic stand. Carefully remove and discard the cleared
solution from each well. Do not touch the beads while removing the solution.
8 Continue to keep the plate or strip tube in the magnetic stand while you dispense 200 µL of
fresh 70% ethanol in each sample well.
9 Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol.
10 Repeat step 8 and step 9 once for a total of two washes.
11 Cap the wells, then briefly spin the samples to collect the residual ethanol. Return the samples
to the magnetic stand for 30 seconds. Remove the residual ethanol with a P20 pipette.

SureSelect Cancer CGP Assay User Guide 21

 12 Dry the samples by placing the unsealed plate or strip tube on the thermal cycler, set to hold
samples at 37°C, until the residual ethanol has just evaporated (up to 2 minutes).
Samples can instead be dried by keeping the unsealed plate or strip tube on the benchtop for
approximately 5 minutes or until the residual ethanol has just evaporated.
Do not dry the bead pellet to the point that the pellet appears cracked. Elution efficiency is
N OTE significantly decreased when the bead pellet is excessively dried.

13 Elute the cDNA by adding 52 µL of nuclease-free water to each sample well.

14 Mix by pipetting up and down 10–15 times or cap the wells and vortex at high speed for
5 seconds. Verify that all beads have been resuspended, with no visible clumps in the
suspension or bead pellets retained on the sides of the wells. If samples were vortexed, spin
briefly to collect the liquid, being careful not to pellet the beads.
15 Incubate for 2 minutes at room temperature.
16 Put the plate or strip tube in the magnetic stand and leave until the solution is clear (up to
5 minutes).
17 Remove 50 µL of cleared supernatant to a fresh PCR plate or strip tube sample well and keep
on ice. You can discard the beads at this time.
The purified cDNA is ready for NGS library preparation; proceed to “Library Preparation and
Pre-capture Amplification” on page 30 to continue processing the cDNA samples. NGS library
preparation from fragmented gDNA samples may be performed in parallel; proceed to page 23 for
gDNA sample processing instructions.
Stopping Point If you do not continue to the next step, seal the wells and store at 4°C overnight or at –20°C for
prolonged storage.

SureSelect Cancer CGP Assay User Guide 22

Agilent SureSelect Cancer CGP Assay
User Guide

3
DNA-Specific Workflow Steps
Step 1. Prepare and qualify the genomic DNA samples 24
FFPE DNA samples 24
Intact DNA samples 25
Step 2. Fragment the DNA 25
Method 1: Enzymatic DNA fragmentation 25
Method 2: Mechanical DNA shearing with Covaris 27

This section describes the steps to prepare input gDNA samples and fragment the input DNA
either by enzymatic fragmentation or by mechanical shearing to a target fragment length suitable
for NGS with 2 x 150 read length. The protocols include conditions for both FFPE-derived gDNA
samples (see page 24) and intact DNA from fresh or fresh-frozen samples (see page 25).
If you are preparing RNA libraries (only), use the RNA sample preparation instructions starting on
page 16 and skip the instructions in this section.
If you are preparing both DNA and RNA libraries in the same run, see page 16 for DNA & RNA
assay workflow synchronization guidelines.

23
Step 1. Prepare and qualify the genomic DNA samples

FFPE DNA samples

The instructions in this section are for FFPE-derived DNA samples. For intact (non-FFPE) DNA
samples, instead follow the instructions on page 25.
Samples are obtained from tissue resection (tissue curls or sections on slide), with use of a
minimum of 3 sections of 5 µm each recommended. FFPE tumor samples should have ≥15%
tumor content (measured by haemotoxylin & eosin staining).

1 Prepare gDNA from FFPE tissue sections using QIAGEN’s QIAamp DNA FFPE Tissue Kit and
Deparaffinization Solution, following the manufacturer’s protocol.
Elute the final gDNA samples from the MinElute column in two rounds, using 30 µL Buffer ATE
in each round, for a final elution volume of approximately 60 µL.
Store the gDNA samples on ice for same-day library preparation, or at –20°C for later
processing.
2 Use the Qubit BR dsDNA Assay Kit to determine the concentration of each gDNA sample.
Follow the manufacturer’s instructions for the instrument and assay kit.
3 Assess the quality (DNA integrity) for each FFPE DNA sample using one of the methods below.

Option 1: Qualification using the Agilent Genomic DNA ScreenTape assay DIN score
The Agilent TapeStation Genomic DNA ScreenTape assay provides a quantitative
electrophoretic assay for DNA sample integrity determination. This assay reports a DNA
Integrity Number (DIN) score for each sample which is used to estimate the appropriate
normalization of DNA input required for low-integrity DNA samples.
a Analyze a 1-µL aliquot of each FFPE gDNA sample using the Genomic DNA ScreenTape
assay. Follow the instructions provided in the assay user manual.
b Consult Table 12 for DIN score-based input DNA input guidelines.
Table 12 DNA input guidelines based on DNA Integrity Number (DIN) score

DIN Score DNA Input Guidelines

DIN > 3 50 ng DNA recommended
DIN 2–3 50 ng DNA required, maximum amount DNA available (up to 200 ng) recommended
DIN <2 Not recommended for further processing

Option 2: Qualification using the Agilent NGS FFPE QC Kit

The Agilent NGS FFPE QC Kit provides a qPCR-based assay for DNA sample integrity
determination. Results include a Cq DNA integrity score and the precise quantity of
amplifiable DNA in the sample, allowing direct normalization of DNA input for each sample.
a Analyze a 1-µL aliquot of each FFPE gDNA sample using the Agilent NGS FFPE QC Kit.
Follow the instructions provided in the kit user manual.
b Consult Table 13 for Cq score-based input DNA input guidelines.
For all samples with Cq DNA integrity score 1 (more intact FFPE DNA samples), use the
Qubit-based gDNA concentration to determine volume of input DNA needed for the
protocol.

SureSelect Cancer CGP Assay User Guide 24

 For all samples with Cq DNA integrity score 1 (less intact FFPE DNA samples), use the
qPCR-based concentration of amplifiable gDNA, reported by the Agilent NGS FFPE QC Kit
results, to determine amounts of input DNA for the protocol.
Table 13 DNA input guidelines based on Cq DNA integrity score

Cq Score DNA Input Guidelines

* 50 ng DNA recommended, quantified by Qubit Assay
Cq1
Cq1 50–200 ng of amplifiable DNA, based on qPCR quantification

* FFPE samples with Cq scores 1should be treated like non-FFPE samples for DNA input amount determinations. For
samples of this type, make sure to use the DNA concentration determined by the Qubit Assay, instead of the concen-
tration determined by qPCR, to calculate the volume required for 50 ng DNA.

Intact DNA samples

1 Prepare high-quality gDNA from fresh or frozen biological samples using a suitable purification
system, such as QIAGEN’s QIAamp DNA Mini Kit, following the manufacturer’s protocol.
Make sure genomic DNA samples are of high quality with an OD 260/280 ratio
N OTE ranging from 1.8 to 2.0.

2 Use the Qubit BR dsDNA Assay Kit to determine the concentration of each gDNA sample.
Follow the manufacturer’s instructions for the instrument and assay kit.
Agilent’s OneSeq Human Reference DNA (supplied at 200 ng/L) is recommended for use as an
intact control DNA sample, which can be included in runs in order to differentiate performance
issues related to sample quality from other factors. When using this intact control DNA sample,
proceed directly to the appropriate DNA fragmentation protocol below for dilution and
fragmentation instructions.

Step 2. Fragment the DNA

In this step the appropriate gDNA samples are fragmented either by enzymatic fragmentation or
by mechanical shearing
The SureSelect XT HS2 DNA system supports use of 10–200 ng DNA input. Use of
N OTE <50 ng DNA for the SureSelect Cancer CGP Assay may reduce yield and target coverage.

Method 1: Enzymatic DNA fragmentation

In this step, gDNA samples are fragmented using Agilent’s SureSelect Enzymatic Fragmentation
Kit. The conditions provided produce fragments suitable for library construction followed by NGS
with 2 x 150 read length.
For FFPE DNA samples, initial DNA fragment size may impact the
N OTE post-fragmentation size distribution, resulting in fragment sizes shorter than the
target range for the recommended 2 x 150-read NGS in some samples. All
samples, including low-integrity FFPE samples, should be incubated at 37°C for
15 minutes to generate fragment ends suitable for library construction.

SureSelect Cancer CGP Assay User Guide 25

 1 In wells of a thermal cycler-compatible strip tube or PCR plate, dilute 50 ng of each gDNA
sample with nuclease-free water to a final volume of 7 µL.
2 Thaw the vial of 5X SureSelect Fragmentation Buffer, vortex, then place on ice.
3 Preprogram a thermal cycler as shown in Table 14; pause until use in step 7.
Table 14 Thermal cycler program for enzymatic fragmentation (10 l vol)

Step Temperature Time

Step 1 37°C 15 minutes

Step 2 65°C 5 minutes

Step 3 4°C Hold

4 Prepare the appropriate volume of fragmentation master mix by combining the reagents in
Table 15. Mix well then spin briefly and keep on ice.

Table 15 Preparation of fragmentation master mix

Reagent Volume for 1 reaction Volume for 8 reactions* Volume for 24 reactions†
(includes excess) (includes excess)

5X SureSelect Fragmentation Buffer 2 µL 18 µL 50 µL

SureSelect Fragmentation Enzyme 1 µL 9 µL 25 µL

Total 3 µL 27 µL 75 µL

* The minimum supported run size for 16-reaction kits is 8 samples per run, with kits containing enough reagents for 2 runs of 8 samples each.
† The minimum supported run size for 96-reaction kits is 24 samples per run, with kits containing enough reagents for 4 runs of 24 samples each.

5 Add 3 µL of the fragmentation master mix to each sample well containing 7 µL of input DNA.
6 Mix well by pipetting up and down 20 times or cap the wells and vortex at high speed for
5–10 seconds. Spin the samples briefly.
7 Immediately place the samples in the thermal cycler and resume the enzymatic fragmentation
program in Table 14.
8 When the program reaches the 4°C Hold step, remove the samples from the thermal cycler,
add 40 µL of nuclease-free water to each sample, and place the samples on ice.
The 50-µL reactions are now ready for NGS sequencing library preparation, beginning with end
repair/dA-tailing. Proceed to “Library Preparation and Pre-capture Amplification” on page 30.
This is not a stopping point in the workflow, and analysis of the
N OTE enzymatically-fragmented samples is not required before they are used for library
preparation. Proceed directly to page 30.

SureSelect Cancer CGP Assay User Guide 26

 Method 2: Mechanical DNA shearing with Covaris
In this step, gDNA samples are sheared using conditions optimized for either high-quality or FFPE
DNA in a 50-µL shearing volume. The conditions provided produce fragments suitable for NGS
with 2 x 150 read length. See Table 16 for shearing parameter guidelines.
Table 16 Covaris shearing parameter summary

Parameter Value

Target fragment size 180 to 250 bp*

Shearing duration for FFPE DNA samples 240 seconds

Shearing duration for intact DNA samples 2 × 120 seconds

* For FFPE DNA samples, initial DNA fragment size may impact the post-fragmentation size distribution, resulting in frag-
ment sizes shorter than the target size range shown here. All FFPE samples, including low-integrity samples, should be
sheared for 240 seconds to generate fragment ends suitable for library construction.

Before you begin, set up the Covaris instrument according to the manufacturer’s instructions.
Allow enough time (typically 30–60 minutes) for instrument degassing before starting the
protocol.
This protocol has been optimized using a Covaris model E220 instrument and the
N OTE 130-l Covaris microTUBE. Consult the manufacturer’s recommendations for use
of other Covaris instruments or sample holders to achieve the same target DNA
fragment size.

1 Prepare the DNA samples for the run by diluting 50 ng of each gDNA sample with 1X Low TE
Buffer (10 mM Tris-HCl, pH 7.5-8.0, 0.1 mM EDTA) to a final volume of 50 µL. Vortex well to
mix, then spin briefly to collect the liquid. Keep the samples on ice.
Do not dilute samples to be sheared using water. Shearing samples in water
N OTE reduces the overall library preparation yield and complexity.

2 Complete the DNA shearing steps below for each gDNA sample.
a Transfer the 50-µL DNA sample into a Covaris microTUBE.
b Spin the microTUBE for 30 seconds to collect the liquid and to remove any bubbles from
the bottom of the tube.
c Secure the microTUBE in the tube holder and shear the DNA with the settings in Table 17.
Table 17 Shear settings for Covaris E-series instrument (SonoLab software v7 or later)

Setting FFPE DNA High-quality DNA

Duty Factor 10% 10%
Peak Incident Power (PIP) 175 175
Cycles per Burst 200 200
Treatment Time 240 seconds 2 × 120 seconds (shear 120 sec, spin 10 sec, vortex 5 sec, spin
10 sec, then repeat full sequence once more retaining the
sample in the microTUBE throughout process)
Bath Temperature 2° to 8° C 2° to 8° C

SureSelect Cancer CGP Assay User Guide 27

 d Transfer the sheared DNA sample (approximately 50 µL) to a 96-well plate or strip tube
sample well. Keep the samples on ice.
e After transferring the DNA sample, spin the microTUBE briefly to collect any residual
sample volume. Transfer any additional collected liquid to the sample well used in step d.
The 50-µL sheared DNA samples are now ready for NGS sequencing library preparation,
beginning with end repair/dA-tailing. Proceed to “Library Preparation and Pre-capture
Amplification” on page 30.
This is not a stopping point in the workflow, and analysis of the sheared samples
N OTE is not required before they are used for library preparation. Proceed directly to
page 30.

SureSelect Cancer CGP Assay User Guide 28

Agilent SureSelect Cancer CGP Assay
User Guide

4
DNA/RNA Workflow Steps: Library Prep and
Hybridization to SureSelect Cancer CGP Assay
Probes
Library Preparation and Pre-capture Amplification 30
Step 1. Prepare the ligation master mix 31
Step 2. Repair and dA-tail the DNA 3' ends 31
Step 3. Ligate the molecular-barcoded adaptor 32
Step 4. Purify libraries using AMPure XP Beads 33
Step 5. Amplify the pre-capture libraries 34
Step 6. Purify amplified libraries using AMPure XP Beads 36
Step 7. QC and quantify the pre-capture libraries 37
Hybridization, Capture and Post-capture Amplification 39
Step 1. Hybridize libraries to the SureSelect Cancer CGP Assay Probe 40
Step 2. Prepare streptavidin beads for capture 42
Step 3. Capture the hybridized libraries 42
Step 4. Amplify the captured libraries 43
Step 5. Purify the final libraries using AMPure XP Beads 44
Step 6. QC and quantify final libraries 46

The first module in this section describes the steps to prepare NGS libraries from gDNA or cDNA
fragments. For each sample to be sequenced, an individual dual-indexed library is prepared. To
process multiple samples, the protocol includes steps for preparation of reagent mixtures with
overage, which are afterward distributed to the DNA library samples. Mixtures for preparation of
8 or 24 samples are shown in tables as examples.
The second module in this section describes hybridization of the prepared gDNA or cDNA libraries
to the appropriate SureSelect Cancer CGP Assay Probe. Target-enriched libraries are then
amplified and analyzed prior to pooling for NGS.

29
Library Preparation and Pre-capture Amplification

This workflow segment uses the components listed in Table 18. Remove the listed reagents from
cold storage, and prepare as directed before use (refer to the Where Used column).

Table 18 Reagents thawed before use in protocol

Storage Location Kit Component Preparative Steps Where Used

Ligation Buffer (purple cap or bottle) Thaw on ice (may require page 31
>20 minutes) then keep
on ice, vortex to mix

For DNA-input samples T4 DNA Ligase (blue cap) Place on ice just before page 31
get reagents from the use, invert to mix
SureSelect XT HS2 End Repair-A Tailing Buffer (yellow cap or bottle) Thaw on ice (may require page 32
Library Preparation Kit for >20 minutes) then keep
ILM (Pre PCR) box, stored on ice, vortex to mix
at –20°C
End Repair-A Tailing Enzyme Mix (orange cap) Place on ice just before page 32
For RNA-input samples use, invert to mix
get reagents from the
SureSelect XT HS2 RNA DNA samples: XT HS2 Adaptor Oligo Mix (white cap) Thaw on ice then keep on page 32
Library Preparation Kit for — ice, vortex to mix
ILM (Pre PCR) box, stored RNA samples: XT HS2 RNA Adaptor Oligo Mix (green cap)
at –20°C Herculase II Fusion DNA Polymerase (red cap) Place on ice just before page 35
use, mix by pipetting

5× Herculase II Buffer with dNTPs (clear cap) Thaw on ice then keep on page 35
ice, vortex to mix

–20°C SureSelect XT HS2 Index Primer Pairs Thaw on ice then keep on page 35
DNA samples: Index Pairs 1-16 (blue + white strips) or ice, vortex to mix
Index Pairs 1-96 (orange plate)
—
RNA samples: Index Pairs 17-32 (black + red strips) or
Index Pairs 97-192 (blue plate)

+4°C DNA samples: SureSelect DNA AMPure XP Beads Equilibrate at room page 33 and
— temperature for at least page 36
RNA samples: SureSelect RNA AMPure XP Beads 30 minutes before use,
vortex to mix

SureSelect Cancer CGP Assay User Guide 30

 Step 1. Prepare the ligation master mix
Prepare the ligation master mix to allow equilibration to room temperature while you are
completing the end repair/dA-tailing step. Leave DNA samples on ice while completing this step.
The Ligation Buffer used in this step is viscous. Make sure to follow the mixing instructions
CAU TIO N in step 1 below.

1 Prepare the appropriate volume of ligation master mix by combining the reagents in Table 19.
Vortex the thawed vial of Ligation Buffer for 15 seconds at high speed just before use. Slowly
pipette the Ligation Buffer into a 1.5-mL tube, ensuring that the full volume is dispensed.
Slowly add the T4 DNA Ligase, rinsing the enzyme tip with buffer solution after addition. Mix
well by slowly pipetting up and down 15–20 times or seal the tube and vortex at high speed for
10–20 seconds. Spin briefly.
Keep at room temperature for 30–45 minutes before use on page 32.

Table 19 Preparation of ligation master mix

Reagent Volume for Volume for 8 reactions* Volume for 24 reactions†

1 reaction (includes excess) (includes excess)

Ligation Buffer (purple cap or bottle) 23 µL 207 µL 598 µL

T4 DNA Ligase (blue cap) 2 µL 18 µL 52 µL

Total 25 µL 225 µL 650 µL

* The minimum supported run size for 16-reaction kits is 8 samples per run, with kits containing enough reagents for 2 runs of 8 samples each.
† The minimum supported run size for 96-reaction kits is 24 samples per run, with kits containing enough reagents for 4 runs of 24 samples each.

Step 2. Repair and dA-tail the DNA 3' ends

The End Repair-A Tailing Buffer used in this step is viscous. Make sure to follow the mixing
CAU TIO N instructions in step 2 and step 3 on page 32.

1 Preprogram a thermal cycler as shown in Table 20; pause until use in step 5.
Table 20 Thermal cycler program for end repair/dA-tailing (70 l vol)

Step Temperature Time

Step 1 20°C 15 minutes

Step 2 72°C 15 minutes

Step 3 4°C Hold

SureSelect Cancer CGP Assay User Guide 31

 2 Vortex the thawed vial of End Repair-A Tailing Buffer for 15 seconds at high speed to ensure
homogeneity. Visually inspect the solution; if any solids are observed, continue vortexing until
all solids are dissolved.
3 Prepare the appropriate volume of dA-tailing master mix by combining the reagents in
Table 21.
Slowly pipette the End Repair-A Tailing Buffer into a 1.5-mL tube, ensuring that the full volume
is dispensed. Slowly add the End Repair-A Tailing Enzyme Mix, rinsing the enzyme tip with
buffer solution after addition. Mix well by pipetting up and down 15–20 times with a pipette set
to at least 80% of the mixture volume, or seal the tube and vortex at high speed for 5–10
seconds. Spin briefly and keep on ice.

Table 21 Preparation of end repair/dA-tailing master mix

Reagent Volume for Volume for 8 reactions Volume for 24 reactions

1 reaction (includes excess) (includes excess)
End Repair-A Tailing Buffer (yellow cap or bottle) 16 µL 144 µL 416 µL

End Repair-A Tailing Enzyme Mix (orange cap) 4 µL 36 µL 104 µL

Total 20 µL 180 µL 520 µL

4 Add 20 µL of the end repair/dA-tailing master mix to each sample well containing 50 µL of DNA
(either fragmented gDNA or purified cDNA fragments). Mix by pipetting up and down
15–20 times using a pipette set to 50 µL or cap the wells and vortex at high speed for
5–10 seconds.
5 Briefly spin the samples, then immediately place the plate or strip tube in the thermal cycler
and resume the thermal cycling program in Table 20.

Step 3. Ligate the molecular-barcoded adaptor

1 Once the thermal cycling program in Table 20 reaches the 4°C Hold step, transfer the samples
to ice. Preprogram the cycler as show in Table 22; pause until use in step 4.
Table 22 Thermal cycler program for ligation (100 l vol)

Step Temperature Time

Step 1 20°C 30 minutes

Step 2 4°C Hold

2 To each end-repaired/dA-tailed DNA sample (approximately 70 µL), add 25 µL of the ligation

master mix that was prepared on page 31 and kept at room temperature. Mix by pipetting up
and down at least 10 times using a pipette set to 70 µL or cap the wells and vortex at high
speed for 5–10 seconds. Briefly spin the samples.
3 Add 5 µL of the appropriate SureSelect Adaptor Oligo Mix to each sample:
• For DNA input libraries–5 µL of XT HS2 Adaptor Oligo Mix (white-capped tube)
• For RNA input libraries–5 µL of XT HS2 RNA Adaptor Oligo Mix (green-capped tube)

SureSelect Cancer CGP Assay User Guide 32

 Mix by pipetting up and down 15–20 times using a pipette set to 70 µL or cap the wells and
vortex at high speed for 5–10 seconds.
Make sure to add the ligation master mix and the Adaptor Oligo Mix to the
N OTE samples in separate addition steps, mixing after each addition, as directed above.

4 Briefly spin the samples, then immediately place the plate or strip tube in the thermal cycler
and resume the thermal cycling program in Table 22.
The AMPure XP Beads used in the next step must be equilibrated to room
N OTE temperature for at least 30 minutes before use.

Step 4. Purify libraries using AMPure XP Beads

Once the thermal cycler program in Table 22 reaches the 4°C hold step, purify the libraries using
room-temperature AmpPure XP Beads.
Critical purification protocol parameters are summarized for experienced users in Table 23. A
video demonstrating the AmpPure XP Bead purification protocol is available at Agilent.com.
(Perform all purification steps in plates or strip tubes as described below; do not transfer samples
to 1.5 mL tubes as shown in the video demonstration.)

Table 23 AMPure XP bead cleanup parameters after adaptor ligation

Parameter Value
Volume of RT AMPure XP bead suspension added to each sample well 80 µL
Final elution solvent and volume 35 µL nuclease-free water
Amount of eluted sample transferred to fresh well Approximately 34 µL

1 Prepare 400 µL of 70% ethanol per sample, plus excess, for use in step 8.
The freshly-prepared 70% ethanol may be used for all purification steps run on
N OTE the same day. Consult the workflow summary on page 10 to determine how
many same-day purification steps will be run.

2 Mix the room-temperature AMPure XP Beads well until homogeneous and consistent in color.
3 Transfer the DNA samples from the thermal cycler to room temperature, then add 80 µL of the
bead suspension to each sample well.
4 Mix by pipetting up and down 15–20 times or cap the wells and vortex at high speed for
5–10 seconds then spin briefly to collect the samples, being careful not to pellet the beads.
5 Incubate the bead suspensions for 5 minutes at room temperature.
6 Put the plate or strip tube into a magnetic separation device. Wait for the solution to clear
(approximately 5 to 10 minutes).
7 Keep the plate or strip tube in the magnetic stand. Carefully remove and discard the cleared
solution from each well. Do not touch the beads while removing the solution.
8 Continue to keep the plate or strip tube in the magnetic stand while you dispense 200 µL of
fresh 70% ethanol in each sample well.

SureSelect Cancer CGP Assay User Guide 33

 9 Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol.
10 Repeat step 8 and step 9 once for a total of two washes.
11 Cap the wells, then briefly spin the samples to collect the residual ethanol. Return the samples
to the magnetic stand for 30 seconds. Remove the residual ethanol with a P20 pipette.
12 Dry the samples by placing the unsealed plate or strip tube on the thermal cycler, set to hold
samples at 37°C, until the residual ethanol has just evaporated (up to 2 minutes).
Samples can instead be dried by keeping the unsealed plate or strip tube on the benchtop for
approximately 5 minutes or until the residual ethanol has just evaporated.
Do not dry the bead pellet to the point that the pellet appears cracked. Elution efficiency is
N OTE significantly decreased when the bead pellet is excessively dried.

13 Elute the library DNA by adding 35 µL of nuclease-free water to each sample well.
14 Mix by pipetting up and down 10–15 times or cap the wells and vortex at high speed for
5 seconds. Verify that all beads have been resuspended, with no visible clumps in the
suspension or bead pellets retained on the sides of the wells. If samples were vortexed, spin
briefly to collect the liquid, being careful not to pellet the beads.
15 Incubate for 2 minutes at room temperature.
16 Put the plate or strip tube in the magnetic stand and leave until the solution is clear (up to
5 minutes).
17 Remove the cleared supernatant (approximately 34 µL) to a fresh PCR plate or strip tube
sample well and keep on ice. You can discard the beads at this time.

Step 5. Amplify the pre-capture libraries

1 Determine the appropriate index pair assignment for each sample. See Table 67 on page 80
through Table 70 on page 83 for nucleotide sequences of the 8 bp index portion of the primers
used to amplify the DNA libraries in this step.
Use a different indexing primer pair for each sample to be sequenced in the same lane.
Agilent’s SureSelect XT HS2 index pairs use a uniform numbering system across
N OTE all platforms and formats. For example, index pairs 1-8 provided in blue strip
tubes in 16-reaction kits are equivalent to index pairs 1-8 provided in orange
plates in 96-reaction kits and to index pairs 1-8 provided in Magnis automation
system XT HS2 black index strips (labeled D1). Do not combine samples indexed
with the same-numbered index pair from different kit formats for multiplex
sequencing.
When using index pairs provided in strip tubes in step 5 on page 35, verify the
strip tube orientation using the numeral (1, 9, 17 or 25) etched adjacent to the
lowest-numbered index and the strip barcode adjacent to the highest-numbered
index. Pierce the foil seal of the appropriate well with a pipette tip just before
pipetting the solution.

The SureSelect XT HS2 Index Primer Pairs are provided in single-use aliquots. To avoid
CAU TIO N cross-contamination of libraries, do not retain and re-use any residual volume for
subsequent experiments.

SureSelect Cancer CGP Assay User Guide 34

 2 Preprogram a thermal cycler as shown in Table 24; pause until use in step 6.

Table 24 Pre-capture PCR thermal cycler program (50 l vol; heated lid ON)

Segment Number of Cycles* Temperature Time

1 1 98°C 2 minutes
2 FFPE DNA input: 12 cycles 98°C 30 seconds
Intact DNA input: 9 cycles
60°C 30 seconds
FFPE RNA input: 15 cycles
Intact RNA input: 12 cycles 72°C 1 minute
3 1 72°C 5 minutes
4 1 4°C Hold

* See Troubleshooting on page 86 for PCR cycle number optimization recommendations for low-input libraries
and for remediation of low-yield libraries.

To avoid cross-contaminating libraries, set up PCR reactions (all components except the
CAU TIO N library DNA) in a dedicated clean area or PCR hood with UV sterilization and positive air
flow.

3 Prepare the appropriate volume of pre-capture PCR reaction mix, as described in Table 25, on
ice. Mix well on a vortex mixer.

Table 25 Preparation of pre-capture PCR reaction mix

Reagent Volume for Volume for 8 reactions Volume for 24 reactions

1 reaction (includes excess) (includes excess)
5× Herculase II Buffer with dNTPs (clear cap) 10 µL 90 µL 260 µL
Herculase II Fusion DNA Polymerase (red cap) 1 µL 9 µL 26 µL
Total 11 µL 99 µL 286 µL

4 Add 11 µL of the PCR reaction mixture prepared in Table 25 to each sample well containing
purified DNA library (34 µL).
5 Add 5 µL of the appropriate SureSelect XT HS2 Index Primer Pair to each reaction.
Cap the wells then vortex at high speed for 5 seconds. Spin the plate or strip tube briefly to
collect the liquid and release any bubbles.
6 Before adding the samples to the thermal cycler, resume the thermal cycling program in
Table 24 to bring the temperature of the thermal block to 98°C. Once the cycler has reached
98°C, immediately place the sample plate or strip tube in the thermal block and close the lid.
The lid of the thermal cycler is hot and can cause burns. Use caution when working near
CAU TIO N the lid.

Stopping Point If you do not continue to the next step, seal the sample wells and store at 4°C overnight or at
–20°C for prolonged storage.
The AMPure XP Beads used in the next step must be equilibrated to room temperature for
N OTE at least 30 minutes before use.

SureSelect Cancer CGP Assay User Guide 35

 Step 6. Purify amplified libraries using AMPure XP Beads
Once the thermal cycler program in Table 24 reaches the 4°C hold step, purify the libraries using
room-temperature AmpPure XP Beads.
Critical purification protocol parameters are summarized for experienced users in Table 26.

Table 26 AMPure XP bead cleanup parameters after pre-capture PCR

Parameter Value
Volume of RT AMPure XP bead suspension added to each sample well 50 µL
Final elution solvent and volume 15 µL nuclease-free water
Amount of eluted sample transferred to fresh well Approximately 14 µL

1 Prepare 400 µL of 70% ethanol per sample, plus excess, for use in step 8.
2 Mix the room-temperature AMPure XP Beads well until homogeneous and consistent in color.
3 Transfer the library DNA samples from the thermal cycler to room temperature, then add 50 µL
of the bead suspension to each sample well.
4 Mix by pipetting up and down 15–20 times or cap the wells and vortex at high speed for
5–10 seconds then spin briefly to collect the samples, being careful not to pellet the beads.
5 Incubate the bead suspensions for 5 minutes at room temperature.
6 Put the plate or strip tube into a magnetic separation device. Wait for the solution to clear
(approximately 5 minutes).
7 Keep the plate or strip tube in the magnetic stand. Carefully remove and discard the cleared
solution from each well. Do not touch the beads while removing the solution.
8 Continue to keep the plate or strip tube in the magnetic stand while you dispense 200 µL of
fresh 70% ethanol in each sample well.
9 Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol.
10 Repeat step 8 and step 9 once for a total of two washes.
11 Cap the wells, then briefly spin the samples to collect the residual ethanol. Return the samples
to the magnetic stand for 30 seconds. Remove the residual ethanol with a P20 pipette.
12 Dry the samples by placing the unsealed plate or strip tube on the thermal cycler, set to hold
samples at 37°C, until the residual ethanol has just evaporated (up to 2 minutes).
Samples can instead be dried by keeping the unsealed plate or strip tube on the benchtop for
approximately 5 minutes or until the residual ethanol has just evaporated.
Do not dry the bead pellet to the point that the pellet appears cracked. Elution efficiency is
N OTE significantly decreased when the bead pellet is excessively dried.

13 Elute the library DNA by adding 15 µL of nuclease-free water to each sample well.
14 Mix by pipetting up and down 10–15 times or cap the wells and vortex at high speed for
5 seconds. Verify that all beads have been resuspended, with no visible clumps in the
suspension or bead pellets retained on the sides of the wells. If samples were vortexed, spin
briefly to collect the liquid, being careful not to pellet the beads.
15 Incubate for 2 minutes at room temperature.

SureSelect Cancer CGP Assay User Guide 36

 16 Put the plate or strip tube in the magnetic stand and leave until the solution is clear (up to
5 minutes).
17 Remove the cleared supernatant (approximately 14 µL) to a fresh PCR plate or strip tube
sample well and keep on ice. You can discard the beads at this time.
Stopping Point If you do not plan to continue through the hybridization step on same day, seal the wells and store
at 4°C overnight or at –20°C for prolonged storage (remove aliquot for QC analysis before
storage, if appropriate).

Step 7. QC and quantify the pre-capture libraries

Analyze a sample of each library using one of the platforms listed in Table 27. Follow the
instructions in the linked user guide provided for each assay.

Table 27 Pre-capture library analysis options

Analysis platform Assay used at this Link to assay instructions Amount of library
step sample to analyze

Agilent 4200/4150 TapeStation D1000 ScreenTape Agilent D1000 Assay Quick 1 µL of five-fold
system Guide dilution

Agilent 2100 Bioanalyzer system DNA 1000 Kit Agilent DNA 1000 Kit Guide 1 µL of five-fold
dilution

Agilent 5200/5300/5400 NGS Fragment Kit Agilent NGS Fragment Kit 2 µL of five-fold
Fragment Analyzer system (1-6000 bp) (1-6000 bp) Kit Guide dilution

Each analysis method provides an electropherogram showing the size distribution of fragments
in the sample and tools for determining the concentration of DNA in the sample. See Table 28 for
fragment size distribution guidelines. Representative electropherograms generated using the
TapeStation system are provided in Figure 2 and Figure 3 to illustrate typical results.

Table 28 Pre-capture library qualification guidelines

Input type Expected library DNA fragment size peak position

FFPE DNA 200 to 400 bp
Intact DNA 270 to 400 bp
RNA (FFPE or Intact) 200 to 700 bp

Observation of a low molecular weight peak, in addition to the expected library fragment peak,
indicates the presence of adaptor-dimers in the library. It is acceptable to proceed to target
enrichment with library samples for which adaptor-dimers are observed in the electropherogram
at low abundance, similar to that seen in example electropherogram in Figure 3. See
Troubleshooting on page 87 for additional considerations.

SureSelect Cancer CGP Assay User Guide 37

 Figure 2 Pre-capture library prepared from an enzymatically fragmented FFPE gDNA sample,
analyzed using a D1000 ScreenTape assay.

Figure 3 Pre-capture library prepared from an FFPE RNA sample, analyzed using a D1000
ScreenTape assay.

Stopping Point If you do not continue to the next step, seal the sample wells and store at 4°C overnight or at
–20°C for prolonged storage.

SureSelect Cancer CGP Assay User Guide 38

Hybridization, Capture and Post-capture Amplification

In this workflow segment, the prepared gDNA libraries are hybridized to the SureSelect Cancer
CGP Assay Probe(s). For each sample library prepared, do one hybridization and capture. The
captured libraries are pooled for multiplexed sequencing after all capture steps are complete.
The hybridization reaction requires 500-1000 ng of prepared library for the DNA assay and 200 ng
of prepared library for the RNA assay, in a volume of 12 µL.
This workflow segment uses the components listed in Table 29. Remove the listed reagents from
cold storage, when required, and prepare as directed before use (refer to the Where Used column).

Table 29 Reagents for Hybridization and Capture

Storage Location Kit Component Preparative Steps Where Used

SureSelect XT HS2 Blocker Mix (blue cap) Thaw and keep on ice, page 40
vortex to mix
SureSelect XT HS2 Target Enrichment
SureSelect RNase Block (purple cap) Thaw and keep on ice, page 40
Kit ILM Hyb Module, Box 2 (Post PCR),
vortex to mix
stored at –20°C
SureSelect Fast Hybridization Buffer (bottle) Thaw and keep at room page 41
temperature

–80°C For DNA-input libraries: SureSelect Cancer CGP Thaw and keep on ice, page 41
Assay Probe DNA (red cap) vortex to mix
—
For RNA-input libraries: SureSelect Cancer CGP
Assay Probe RNA (blue cap)

+4°C SureSelect Streptavidin Beads (clear cap or Remove from 4°C just page 42
bottle) before use, vortex to mix

SureSelect Binding Buffer (bottle) Ready to use page 42

SureSelect Target Enrichment Kit, ILM
Hyb Module, Box 1 (Post PCR), stored SureSelect Wash Buffer 1 (bottle) Ready to use page 42
at RT
SureSelect Wash Buffer 2 (bottle) Ready to use page 42

Herculase II Fusion DNA Polymerase (red cap) Place on ice just before page 44
use, pipette to mix
SureSelect XT HS2 Target Enrichment
5× Herculase II Buffer with dNTPs (clear cap) Thaw and keep on ice, page 44
Kit ILM Hyb Module, Box 2 (Post PCR),
vortex to mix
stored at –20°C
SureSelect Post-Capture Primer Mix (clear cap) Thaw and keep on ice, page 44
vortex to mix

+4°C For DNA-input libraries: SureSelect DNA Equilibrate at room page 44

AMPure XP Beads (bottle) temperature for at least
— 30 minutes before use,
For RNA-input libraries: SureSelect RNA vortex to mix
AMPure XP Beads (bottle)

SureSelect Cancer CGP Assay User Guide 39

 Step 1. Hybridize libraries to the SureSelect Cancer CGP Assay Probe
1 Preprogram a thermal cycler as shown in Table 30; pause until samples are loaded in step 4.

Table 30 Pre-programmed thermal cycler program for hybridization (30 l vol; heated lid ON)

Segment Number Temperature Time

Number of Cycles
1 1 95°C 5 minutes
2 1 65°C 10 minutes
3 1 65°C 1 minute (Pause cycler here for reagent addition, see step 7 on page 41)
65°C 1 minute
4 60
37°C 3 seconds
5 1 65°C Hold briefly until ready to begin capture steps on page 42

2 Place 1000 ng of each prepared gDNA library or 200 ng of each cDNA library (prepared from
RNA samples) into the hybridization plate or strip tube wells. Bring the final volume in each
well to 12 µL using nuclease-free water.
If 1000 ng gDNA library is not available for any of the DNA assay samples, use the maximum
amount of library available, within the 500–1000 ng range.
3 To each DNA library sample well, add 5 µL of SureSelect XT HS2 Blocker Mix (blue cap). Seal
the wells then vortex at high speed for 5 seconds. Spin briefly to collect the liquid and release
any bubbles.
The lid of the thermal cycler is hot and can cause burns. Use caution when working near
CAU TIO N the lid.

4 Transfer the sealed sample plate or strip to the thermal cycler and resume the thermal cycling
program in Table 30, allowing the cycler to complete Segments 1 and 2 of the program.
Important: The thermal cycler must be paused during Segment 3 to allow additional
reagents to be added to the Hybridization wells, as described in step 7 on page 41.
During Segments 1 and 2 of the thermal cycling program, begin preparing the additional
hybridization reagents as described in step 5 below and step 6 on page 41. If needed, you can
finish these preparation steps after pausing the thermal cycler in Segment 3.
5 Prepare a 25% solution of SureSelect RNase Block (1 part RNase Block to 3 parts water)
according to Table 31. Prepare the amount required for the number of hybridization reactions
in the run, plus excess. Mix well and keep on ice.
Table 31 Preparation of RNase Block solution

Reagent Volume for 1 reaction Volume for 8 reactions Volume for 24 reactions
(includes excess) (includes excess)
SureSelect RNase Block 0.5 µL 4.5 µL 12.5 µL
Nuclease-free water 1.5 µL 13.5 µL 37.5 µL
Total 2 µL 18 µL 50 µL

SureSelect Cancer CGP Assay User Guide 40

 Prepare the mixture described in step 6, below, just before pausing the thermal
N OTE cycler in Segment 3. Keep the mixture at room temperature briefly until the
mixture is added to the DNA samples in step 7. Do not keep solutions containing
the probe at room temperature for extended periods.

6 Prepare the probe hybridization mix according to Table 32. Combine the listed reagents at
room temperature. Mix well by vortexing at high speed for 5 seconds then spin down briefly.
Proceed immediately to step 7.
Table 32 Preparation of probe hybridization mix

Reagent Volume for 1 Volume for 8 reactions Volume for 24 reactions

reaction (includes excess) (includes excess)

25% RNase Block solution (from step 5) 2 µL 18 µL 50 µL

SureSelect Cancer CGP Assay Probe 2 µL 18 µL 50 µL

(DNA probe OR RNA probe)*
SureSelect Fast Hybridization Buffer 6 µL 54 µL 150 µL

Nuclease-free water 3 µL 27 µL 75 µL

Total 13 µL 117 µL 325 µL

* Add either SureSelect Cancer CGP Assay Probe DNA OR SureSelect Cancer CGP Assay Probe RNA; do not combine DNA and RNA assay
probes in the same hybridization reaction.

7 Once the thermal cycler starts Segment 3 (1 minute at 65°C), pause the program. With the
cycler paused, and while keeping the DNA + Blocker samples in the cycler, transfer 13 µL of the
room-temperature probe hybridization mix from step 6 to each sample well.
Mix well by pipetting up and down slowly 8 to 10 times.
The hybridization reaction wells now contain approximately 30 µL.
8 Seal the wells with fresh strip caps. Make sure that all wells are completely sealed. Vortex
briefly, then spin the plate or strip tube briefly to remove any bubbles from the bottom of the
wells. Immediately return the plate or strip tube to the thermal cycler.
9 Resume the thermal cycling program to allow hybridization of the prepared library DNA
samples to the probe.
Wells must be adequately sealed to minimize evaporation, or your results can be
CAU TIO N negatively impacted.
Before you do the first experiment, make sure the plasticware and capping
method are appropriate for the thermal cycler. Check that no more than 4 µL is
lost to evaporation under the conditions used for hybridization.

SureSelect Cancer CGP Assay User Guide 41

 Step 2. Prepare streptavidin beads for capture
1 Vigorously resuspend the vial of SureSelect Streptavidin Beads on a vortex mixer. The
magnetic beads settle during storage.
2 For each hybridization sample, add 50 µL of the resuspended beads to wells of a fresh PCR
plate or strip tube.
3 Wash the beads:
a Add 200 µL of SureSelect Binding Buffer per well of beads.
b Mix by pipetting up and down 20 times or cap the wells and vortex at high speed for
5–10 seconds then spin down briefly.
c Put the plate or strip tube into a magnetic separator device.
d Wait 5 minutes or until the solution is clear, then remove and discard the supernatant.
e Repeat step a through step d two more times for a total of 3 washes.
4 Resuspend the beads in 200 µL of SureSelect Binding Buffer.
If you are equipped for higher-volume magnetic bead captures, the streptavidin
N OTE beads may instead be batch-washed in an Eppendorf tube or conical vial.

Step 3. Capture the hybridized libraries

1 After all streptavidin bead preparation steps are complete, and once the hybridization thermal
cycling program reaches the 65°C hold step (Segment 5; see Table 30 on page 40), transfer
the samples to room temperature.
2 Immediately transfer the entire volume (approximately 30 µL) of each hybridization mixture to
wells containing 200 µL of washed streptavidin beads using a multichannel pipette.
Pipette up and down 5–8 times to mix then seal the wells with fresh caps.
3 Incubate the capture plate or strip tube on a 96-well plate mixer, mixing vigorously (at
1400–1900 rpm), for 30 minutes at room temperature.
Make sure the samples are properly mixing in the wells.
4 During the 30-minute incubation for capture, prewarm SureSelect Wash Buffer 2 at 70°C as
described below.
a Place 200-µL aliquots of Wash Buffer 2 in wells of a fresh 96-well plate or strip tubes.
Aliquot 6 wells of buffer for each DNA sample in the run.
b Cap the wells and then incubate in the thermal cycler held at 70°C until used in step 9.
5 When the 30-minute capture incubation period initiated in step 3 is complete, spin the samples
briefly to collect the liquid.
6 Put the plate or strip tube in a magnetic separator to collect the beads. Wait until the solution
is clear (approximately 1 to 2 minutes), then remove and discard the supernatant.
7 Resuspend the beads in 200 µL of SureSelect Wash Buffer 1. Mix by pipetting up and down
15–20 times, until beads are fully resuspended.
8 Put the plate or strip tube in the magnetic separator. Wait for the solution to clear
(approximately 1 minute), then remove and discard the supernatant.

SureSelect Cancer CGP Assay User Guide 42

 It is important to maintain bead suspensions at 70°C during the washing
CAU TIO N procedure below to ensure specificity of capture.
Make sure that the SureSelect Wash Buffer 2 is pre-warmed to 70°C before use.
Do not use a tissue incubator, or other devices with significant temperature
fluctuations, for the incubation steps.

9 Remove the plate or strip tubes from the magnetic separator and transfer to a rack at room
temperature. Wash the beads with Wash Buffer 2, using the steps below.
a Resuspend the beads in 200 µL of 70°C prewarmed Wash Buffer 2. Pipette up and down
15–20 times, until beads are fully resuspended.
b Seal the wells with fresh caps and then vortex at high speed for 8 seconds. Spin the plate or
strip tube briefly to collect the liquid without pelleting the beads.
Make sure the beads are in suspension before proceeding.
c Incubate the samples for 5 minutes at 70°C on the thermal cycler with the heated lid on.
d Put the plate or strip tube in the magnetic separator at room temperature.
e Wait 1 minute for the solution to clear, then remove and discard the supernatant.
f Repeat step a through step e five more times for a total of 6 washes.
10 After verifying that all wash buffer has been removed, add 25 µL of nuclease-free water to
each sample well. Pipette up and down 8 times to resuspend the beads.
11 Keep the samples on ice until they are used in the PCR reactions below.
Captured DNA is retained on the streptavidin beads during the post-capture
N OTE amplification step.

Step 4. Amplify the captured libraries

1 Preprogram a thermal cycler as shown in Table 33; pause until use in step 5.

Table 33 Post-Capture PCR thermal cycler program (50 l vol; heated lid ON)

Segment Number of Cycles Temperature Time

1 1 98°C 2 minutes
2 13 98°C 30 seconds
60°C 30 seconds
72°C 1 minute
3 1 72°C 5 minutes
4 1 4°C Hold

SureSelect Cancer CGP Assay User Guide 43

 2 Prepare the appropriate volume of post-capture PCR reaction mix, as described in Table 34, on
ice. Mix well on a vortex mixer.

Table 34 Preparation of post-capture PCR reaction mix

Reagent Volume for Volume for 8 reactions Volume for 24 reactions

1 reaction (includes excess) (includes excess)
Nuclease-free water 13 µL 117 µL 338 µL
5× Herculase II Buffer with dNTPs (clear cap) 10 µL 90 µL 260 µL
Herculase II Fusion DNA Polymerase (red cap) 1 µL 9 µL 26 µL
SureSelect Post-Capture Primer Mix (clear cap) 1 µL 9 µL 26 µL
Total 25 µL 225 µL 650 µL

3 Add 25 µL of the PCR reaction mix prepared in Table 34 to each sample well containing 25 µL
of bead-bound target-enriched DNA.
4 Mix the PCR reactions well by pipetting up and down until the bead suspension is
homogeneous. Avoid splashing samples onto well walls; do not spin the samples at this step.
5 Place the plate or strip tube in the thermal cycler and resume the thermal cycling program in
Table 33.
6 When the PCR amplification program is complete, spin the plate or strip tube briefly. Remove
the streptavidin beads by placing the plate or strip tube on the magnetic stand at room
temperature. Wait 2 minutes for the solution to clear, then remove each supernatant
(approximately 50 µL) to wells of a fresh plate or strip tube.
The streptavidin beads can be discarded at this time.
Stopping Point If you do not continue to the next step, seal the sample wells and store at 4°C overnight or at
–20°C for prolonged storage.
The AMPure XP Beads used in the next step must be equilibrated to room temperature for
N OTE at least 30 minutes before use.

Step 5. Purify the final libraries using AMPure XP Beads

Purify the amplified libraries using room-temperature AmpPure XP Beads. Critical purification
protocol parameters are summarized for experienced users in Table 35.

Table 35 AMPure XP bead cleanup parameters after post-capture PCR

Parameter Value
Volume of RT AMPure XP bead suspension added to each sample well 50 µL
Final elution solvent and volume 25 µL Low TE Buffer
Amount of eluted sample transferred to fresh well Approximately 24 µL

1 Prepare 400 µL of 70% ethanol per sample, plus excess, for use in step 8.
2 Mix the room-temperature AMPure XP Beads well until homogeneous and consistent in color.

SureSelect Cancer CGP Assay User Guide 44

 3 Add 50 µL of the bead suspension to each amplified DNA sample (approximately 50 µL) in the
PCR plate or strip tube well.
4 Mix by pipetting up and down 15–20 times or cap the wells and vortex at high speed for
5–10 seconds then spin briefly to collect the samples, being careful not to pellet the beads.
5 Incubate the bead suspensions for 5 minutes at room temperature.
6 Put the plate or strip tube into a magnetic separation device. Wait for the solution to clear
(approximately 2 to 5 minutes).
7 Keep the plate or strip tube in the magnetic stand. Carefully remove and discard the cleared
solution from each well. Do not touch the beads while removing the solution.
8 Continue to keep the plate or strip tube in the magnetic stand while you dispense 200 µL of
fresh 70% ethanol in each sample well.
9 Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol.
10 Repeat step 8 and step 9 once for a total of two washes.
11 Cap the wells, then briefly spin the samples to collect the residual ethanol. Return the samples
to the magnetic stand for 30 seconds. Remove the residual ethanol with a P20 pipette.
12 Dry the samples by placing the unsealed plate or strip tube on the thermal cycler, set to hold
samples at 37°C, until the residual ethanol has just evaporated (up to 2 minutes).
Samples can instead be dried by keeping the unsealed plate or strip tube on the benchtop for
approximately 5 minutes or until the residual ethanol has just evaporated.
Do not dry the bead pellet to the point that the pellet appears cracked. Elution efficiency is
N OTE significantly decreased when the bead pellet is excessively dried.

13 Elute the library DNA by adding 25 µL of Low TE buffer to each sample well.
14 Mix by pipetting up and down 10–15 times or cap the wells and vortex at high speed for
5 seconds. Verify that all beads have been resuspended, with no visible clumps in the
suspension or bead pellets retained on the sides of the wells. If samples were vortexed, spin
briefly to collect the liquid, being careful not to pellet the beads.
15 Incubate for 2 minutes at room temperature.
16 Put the plate or strip tube in the magnetic stand and leave until the solution is clear (up to
5 minutes).
17 Remove the cleared supernatant (approximately 24 µL) to a fresh PCR plate or strip tube
sample well and keep on ice. You can discard the beads at this time.
Stopping Point If you do not plan to continue through the library pooling for NGS step on same day, seal the wells
and store at 4°C overnight or at –20°C for prolonged storage (remove aliquot for QC analysis
before storage, if appropriate).

SureSelect Cancer CGP Assay User Guide 45

 Step 6. QC and quantify final libraries
Analyze a sample of each library using one of the platforms listed in Table 36. Follow the
instructions in the linked user guide provided for each assay.

Table 36 Post-capture library analysis options

Analysis platform Assay used at this Link to assay instructions Amount of library
step sample to analyze

Agilent 4200/4150 TapeStation High Sensitivity Agilent High Sensitivity 2 µL

system D1000 ScreenTape D1000 Assay Quick Guide

Agilent 2100 Bioanalyzer system High Sensitivity Agilent High Sensitivity 1 µL

DNA Kit DNA Quick Guide

Agilent 5200/5300/5400 HS NGS Fragment Agilent HS NGS Fragment 2 µL

Fragment Analyzer system Kit (1-6000 bp) Kit (1-6000 bp) Kit Guide

Each analysis method provides an electropherogram showing the size distribution of fragments
in the sample and tools for determining the concentration of DNA in the sample. See Table 37 for
fragment size distribution guidelines. Representative electropherograms generated using the
TapeStation system are provided in Figure 4 and Figure 5 to illustrate typical results.

Table 37 Post-capture library qualification guidelines

Input type Expected library DNA fragment size peak position

DNA (FFPE or intact) 200 to 450 bp
RNA (FFPE or Intact) 200 to 700 bp

Figure 4 Post-capture library prepared from an enzymatically fragmented FFPE gDNA sample,
analyzed using a High Sensitivity D1000 ScreenTape assay.

SureSelect Cancer CGP Assay User Guide 46

 Figure 5 Post-capture library prepared from an FFPE RNA sample, analyzed using a High Sensi-
tivity D1000 ScreenTape assay.

Stopping Point If you do not continue to the next step, seal the sample wells and store at 4°C overnight or at
–20°C for prolonged storage.

SureSelect Cancer CGP Assay User Guide 47

Agilent SureSelect Cancer CGP Assay
User Guide

5
NGS and Analysis Workflow Steps
Step 1. Pool samples for multiplexed sequencing 49
Step 2. Prepare the sequencing samples 50
Step 3. Sequence the libraries 51
Step 4. Process the reads to analysis-ready files 52
Step 5. Analyze using Alissa Reporter software 53
Analysis Considerations 57

This section provides guidelines for the NGS and analysis segments of the workflow.
The SureSelect Cancer CGP libraries are sequenced using standard Illumina paired-end primers
and chemistry. The sequencing parameters below are recommended for optimal SureSelect
Cancer CGP Assay analysis performance:
• Depth of ≥40M reads per sample for SureSelect Cancer CGP DNA Assay samples
• Depth of ≥10M reads per sample for SureSelect Cancer CGP RNA Assay samples
• Read length of 2 × 150 bp (recommended for optimal translocation detection in DNA samples)
After reads are demultiplexed, Agilent’s Alissa Reporter software provides a complete
FASTQ-to-Report solution for the SureSelect Cancer CGP assays, processing NGS data from
FASTQ format to VCF format, and reporting SNV, InDel, CNV, translocation, RNA fusion and RNA
exon skipping calls along with TMB and MSI values. Alternatively, the demultiplexed reads can be
pre-processed using Agilent’s Genomics NextGen Toolkit (AGeNT) and the processed reads
analyzed using the appropriate variant analysis tools.

48
Step 1. Pool samples for multiplexed sequencing

The number of indexed libraries that may be multiplexed in a single sequencing lane is
determined by the output specifications of the sequencer used, together with the amount of
sequencing data required per sample (≥40M reads for each DNA sample and ≥10M reads for RNA
samples). If you wish to sequence DNA and RNA libraries together in the same lane, first make
separate pools for RNA and DNA samples at the same concentration (e.g., 10 nM in each pool),
then combine the RNA and DNA pools at 4 parts DNA pool to 1 part RNA pool.
Combine the libraries such that each indexed library is present in equimolar amounts in the pool
using one of the following methods. Use the diluent specified by your sequencing provider, such
as Low TE, for the dilution steps.
Method 1: Dilute each library sample to be pooled to the same final concentration (typically
4–15 nM, or the concentration of the most dilute sample) then combine equal volumes of all
samples to create the final pool.
Method 2: Starting with library samples at different concentrations, add the appropriate volume of
each sample to achieve equimolar concentration in the pool, then adjust the pool to the desired
final volume using Low TE. The formula below is provided for determination of the amount of
each indexed sample to add to the pool.

Vf  Cf
Volume of Index = --------------------------------
#  Ci

where V(f) is the final desired volume of the pool,

C(f) is the desired final concentration of all the DNA in the pool (typically 4 nM–15 nM or the
concentration of the most dilute sample)
# is the number of indexes, and
C(i) is the initial concentration of each indexed sample

Table 38 shows an example of the amount of 4 index-tagged samples (of different

concentrations) and Low TE needed for a final volume of 20 µL at 10 nM DNA.
Table 38 Example of volume calculation for total volume of 20 µL at 10 nM concentration

Component V(f) C(i) C(f) # Volume to use (µL)

Sample 1 20 µL 20 nM 10 nM 4 2.5
Sample 2 20 µL 10 nM 10 nM 4 5
Sample 3 20 µL 17 nM 10 nM 4 2.9
Sample 4 20 µL 25 nM 10 nM 4 2
Low TE 7.6

If you are sequencing DNA and RNA libraries together in the same lane, first make separate pools
for RNA and DNA samples at the same concentration (e.g., 10 nM) using either of the methods
described above, then combine the RNA and DNA pools at 4 parts DNA pool to 1 part RNA pool.
If you store the library pool before sequencing, add Tween 20 to 0.1% v/v and store at –20°C short
term, or store under the conditions specified by your sequencing provider.

SureSelect Cancer CGP Assay User Guide 49

Step 2. Prepare the sequencing samples

The final SureSelect Cancer CGP library pool is ready for sequencing using standard Illumina
paired-end primers and chemistry. Each fragment in the prepared library contains one target
insert surrounded by sequence motifs required for multiplexed sequencing using the Illumina
platform, as shown in Figure 6.

Figure 6 Content of SureSelect Cancer CGP sequencing library. Each fragment contains one
target insert (blue) surrounded by the Illumina paired-end sequencing elements
(black), unique dual sample indexes (red and green), duplex molecular barcodes
(brown) and the library PCR primers (yellow).

Proceed to cluster amplification using the appropriate Illumina Paired-End Cluster Generation Kit
and sequence the libraries using an Illumina instrument. Table 39 provides guidelines for use of
several instrument and chemistry combinations suitable for this application, including kit
configurations compatible with the recommended 2 × 150 bp read length and seeding
concentration recommendations. For other Illumina NGS platforms, consult Illumina’s
documentation for kit configuration and seeding concentration guidelines.
Follow Illumina’s recommendation for a PhiX control in a low-concentration spike-in for improved
sequencing quality control.
Table 39 Illumina kit configuration selection guidelines

Platform Run Type Read Length SBS Kit Configuration Chemistry Seeding
Concentration
NextSeq 500/550 All Runs 2 × 150 bp 300 Cycle Kit v2.5 1.2–1.5 pM
NextSeq 2000 All Runs 2 × 150 bp 300 Cycle Kit v1, v2, or v3 1000 pM
HiSeq 4000 All Runs 2 × 150 bp 300 Cycle Kit v1 300–400 pM
NovaSeq 6000 Standard Workflow Runs 2 × 150 bp 300 Cycle Kit v1.0 or v1.5 300–600 pM
NovaSeq 6000 Xp Workflow Runs 2 × 150 bp 300 Cycle Kit v1.0 or v1.5 200–400 pM

SureSelect Cancer CGP Assay User Guide 50

Step 3. Sequence the libraries

Set up the sequencing run to generate Read 1 and Read 2 FASTQ files for each sample using the
instrument’s software in standalone mode or using an Illumina run management tool such as
Local Run Manager (LRM), Illumina Experiment Manager (IEM) or BaseSpace. Enter the
appropriate Cycles or Read Length value for your library read length and using 8-bp dual index
reads. See Table 40 showing example settings for 2x150 bp sequencing.
Table 40 Run settings for 2x150 bp sequencing

Run Segment Cycles/Read Length

Read 1 151*
Index 1 (i7) 8

Index 2 (i5) 8

Read 2 151*

* Follow Illumina’s recommendation to add one (1) extra cycle to the desired read length.

Follow Illumina’s instructions for each platform and setup software option, incorporating the
additional setup guidelines below:
• Each of the sample-level indexes (i7 and i5) requires an 8-bp index read. For complete index
sequence information, see page 79.
• No custom primers are used for SureSelect XT HS2 library sequencing. Leave all Custom
Primers options for Read 1, Read 2, Index 1 and Index 2 primers cleared/deselected during run
setup.
• Turn off any adaptor trimming tools included in Illumina’s run setup and read processing
software applications. Adaptors are trimmed in later processing steps using Agilent software
tools to ensure proper processing of the adaptors, including the degenerate molecular
barcodes (MBCs) in the adaptor sequences.
• For runs set up using Illumina’s LRM, IEM, or BaseSpace applications, refer to Illumina’s
instructions and support resources for setting up runs with custom library prep kits and index
kits in the selected software. For use in these applications, the SureSelect XT HS2 index
sequences provided in Table 67 through Table 70 should be converted to .tsv/.csv file format
or copied to a Sample Sheet according to Illumina’s specifications for each application. If you
need assistance with SureSelect XT HS2 run setup in your selected application (e.g.,
generating index files or Sample Sheet templates), contact the SureSelect support team (see
page 2) or your local representative.
• If you will use Agilent’s Alissa Reporter for downstream analysis, sequence filename
requirements and other sequencing setup information is available in the Alissa Reporter
software Help, accessed from the Upload run data screen by clicking the Help icon (?) in the
top right corner. If supported by your sequencer, samples may be split across different flow
cell lanes when required. Alissa Reporter supports automatic merging of FASTQ files for
samples sequenced in multiple lanes.

SureSelect Cancer CGP Assay User Guide 51

Step 4. Process the reads to analysis-ready files

Guidelines for sequencing data processing options are outlined below.

1 Generate demultiplexed FASTQ files for each sample using Illumina’s bcl2fastq, BCL Convert
or DRAGEN software. This process generates paired-end reads based on the dual indexes and
removes sequences with incorrectly paired P5 and P7 indexes. Do not use the MBC/UMI
trimming options offered in Illumina’s demultiplexing software.
If you are using Agilent’s Alissa Reporter software for variant discovery, no further read
processing is required prior to FASTQ file uploads. Proceed to page 53. If using other
analysis software tools, proceed to step 2 below.
2 Complete the FASTQ to BAM pre-processing steps below before analyzing the reads using
non-Agilent analysis software.
The steps below use Agilent’s Genomics NextGen Toolkit (AGeNT), a set of Java-based
software modules for MBC pre-processing, adaptor trimming and duplicate read identification.
This toolkit is designed to enable building, integrating, maintaining, and troubleshooting
internal analysis pipelines for users with bioinformatics expertise. For additional information
and to download this toolkit, visit the AGeNT page at www.agilent.com and review the AGeNT
Best Practices document for processing steps suitable for XT HS2 libraries.
a Remove sequencing adaptors from the reads and extract the MBC sequences using the
AGeNT Trimmer module.
Library fragments include a degenerate molecular barcode (MBC) in each strand (see
Figure 6 on page 50). The MBC sequence and dark bases are located at the 5’ end of both
Read 1 and Read 2. Adaptor trimming should be performed using AGeNT Trimmer module.
Other adaptor trimmers may fail to remove the MBC sequences from the opposite adaptor,
which can affect alignment quality.
If your sequence analysis pipeline excludes MBCs, you can remove the first 5
N OTE bases from Read 1 and Read 2 by masking or trimming before proceeding to
downstream analysis.
If demultiplexing using bcl2fastq, MBCs may be masked by including the base
mask N5Y*,I8,I8,N5Y* (where * is replaced with the remaining read length after
subtracting the 5 masked bases, e.g., use N5Y146,I8,I8,N5Y146 for 2x150 NGS
set up as shown in Table 40 on page 51). The sum of the values following N and
Y must match the read length value in the RunInfo.xml file.
If demultiplexing using BCL Convert, MBCs may be trimmed by including the
following string in the sample sheet header: OverrideCycles,N5Y*;I8;I8;N5Y*
(where * is replaced with read length after trimming, e.g., use
N5Y146;I8;I8;N5Y146 for 2x150 NGS set up as shown in Table 40 on page 51).
The sum of the values following N and Y must match the read length value in the
RunInfo.xml file.
Alternatively, the first 5 bases may be trimmed from the demultiplexed FASTQ
files using a suitable processing tool of your choice, such as seqtk. The AGeNT
Trimmer module can also be used to remove the MBCs while trimming adaptor
sequences. Non-Agilent adaptor trimmers will fail to remove the MBC sequences
from the opposite adaptor which may affect alignment quality.

SureSelect Cancer CGP Assay User Guide 52

 b For DNA assay libraries: Align the trimmed reads and add MBC tags to the aligned BAM files
using a suitable tool such as BWA- MEM. Then use the AGeNT CReaK (Consensus Read
Kit) tool to generate consensus reads and mark or remove duplicates.
For RNA assay libraries: Aligned the trimmed reads using a suitable RNA data alignment
tool. Once alignment is complete, the AGeNT CReaK (Consensus Read Kit) tool can be used
in the single- strand consensus mode to generate consensus reads and mark or remove
duplicates.
The resulting BAM files are ready for downstream analysis including gene expression and
variant discovery. Additional resources for analysis pipeline steps are provided below.

Obtaining BED files: Browser extensible data (BED) files detailing the annotated
coordinates of genomic regions included in the SureSelect Cancer CGP probes are available at
Agilent’s SureDesign site. A targets.txt file listing the genes targeted is also available for each
probe.

RNA library strandedness guidelines: The SureSelect XT HS2 RNA sequencing library
preparation method preserves RNA strandedness using dUTP second-strand marking. The
sequence of read 1, starting at P5 end, matches the reverse complement of the poly-A RNA
transcript strand. Read 2, starting at P7 end, matches the poly-A RNA transcript strand. When
running analysis of this data to determine strandedness, it is important to include this
information. For example, when using the Picard tools (https://broadinstitute.github.io/picard)
to calculate RNA sequencing metrics, it is important to include the parameter
STRAND_SPECIFICITY=SECOND_READ_TRANSCRIPTION_STRAND to correctly calculate the
strand specificity metrics.

Step 5. Analyze using Alissa Reporter software

Alternative NGS analysis software tools can also be used for variant discovery.
N OTE Complete the FASTQ file pre-processing steps on page 52 before analysis using
any non-Agilent software tools. Consult the software documentation for file
upload parameters and analysis settings appropriate for your research goals.

Agilent’s Alissa Reporter analysis software is designed to perform read trimming, alignment of
reads to the reference genome, and variant calling for SureSelect Cancer CGP Assay sequencing
data, displaying pre-set thresholds in the QC dashboard which are fine-tuned to the SureSelect
Cancer CGP assay. This section provides guidelines on how to upload the sample files and set up
analysis. See the appropriate Alissa Reporter software Help topic for more detailed information.
Uploading and analysis of sequencing files in Alissa Reporter can be fully
N OTE automated using Amazon Web Services (AWS) S3. See the Alissa Reporter
Amazon Web Services Technical Guide for more information.

SureSelect Cancer CGP Assay User Guide 53

 1 Upload the set of FASTQ files (format .fastq or .fastq.gz) for the run to the Alissa Reporter
analysis software. From the software Home page, click Upload a new run. Forward and reverse
reads from the same sample must be uploaded in the same run. Cancer CGP DNA and RNA
Assay samples may be uploaded in the same run.
The file upload wizard provides guidance on run and sample attributes entered during file
uploads. Values appropriate for required sample attribute fields are summarized in Table 41.
The run setup process can be automated using a meta information manifest file
N OTE (attribute.yaml) that is uploaded with the FASTQ files and is used to pre-populate
the settings in the upload wizard. After adding the attribute.yaml file to the run,
click Parse attribute file to parse the file data and confirm compatibility. For more
information on automated setup including link to an attribute.yaml file template,
click the Help icon (?) on the Upload run data page.

Sample attribute guidelines: Each Cancer CGP DNA Assay tumor sample sequence file to be
analyzed must be designated as a Target sample type during file upload. Each reference DNA
sample sequence file must be designated as either a Matched reference sample or an Unmatched
reference sample during file upload. SureSelect Cancer CGP RNA Assay tumor samples do not
need to be designated as target samples or associated with reference samples for analysis.

Table 41 Alissa Reporter sample file upload settings for the SureSelect Cancer CGP Assays

Field or menu Affected Samples Value

Application All samples Cancer CGP DNA OR Cancer CGP RNA

Application chemistry All samples XTHS2

Sample type Cancer CGP DNA Select Target sample for the experimental tumor sample DNA. Select Matched
samples only reference sample* for a normal reference DNA sample from same individual OR
Unmatched reference sample† for a normal reference DNA sample from another
source (e.g., Agilent’s OneSeq Human Reference DNA)

Reference sample Cancer CGP DNA For each Target sample in the run, expand the Reference sample menu and
samples select the appropriate reference sample name. Matched reference sample files
designated as must be uploaded in the same run as the corresponding Target sample files.
Target sample only Unmatched reference sample files can be uploaded in the same run or a reserved
unmatched reference can be assigned from a previously uploaded run. If the
Target sample will be analyzed without a reference sample, select No reference
sample from the menu. Use of a reference sample is required for CNV calling.

In silico filter Cancer CGP DNA To display analysis results for the full Cancer CGP Probe design, without filtration
Target samples and for specific genes or regions, retain the default setting of No filter. Customized in
Cancer CGP RNA silico filters may be created to restrict displayed analysis results to a selected
samples (not panel of genes and/or genomic regions. For more information on how to set up
entered for DNA and use customized filters, click the Help icon (?) on the in silico filter selection
Reference samples) screen.

Sex All samples Select the sex (X and Y chromosome composition) for each sample. It is
important to use Target samples and Reference samples of the same sex to allow
CNV calling and tumor-normal SNV/Indel calling on the sex chromosomes.

* Each Matched reference sample must be associated with the corresponding Target sample in the same Alissa Reporter run upload.
† Each Unmatched reference sample must be associated with at least one Target sample in the same run upload. Once uploaded the Unmatched
reference sample may also be associated with additional Target samples in later-uploaded runs. To make an unmatched reference sample avail-
able for use in later-uploaded runs, the sample must be reserved by setting the Reserved status of the sample to Yes after uploading.

SureSelect Cancer CGP Assay User Guide 54

 2 Select the appropriate analysis options for the run. Guidelines for key analysis settings are
provided in Table 42 for the Cancer CGP DNA Assay and in Table 43 for the Cancer CGP RNA
Assay. Information on each analysis setting can be obtained during run setup or analysis setup
by clicking the Help icon (?) next to each setting field.
Analysis option settings may also be entered or changed after the sequencing
N OTE files for the run are uploaded.

Table 42 Key Alissa Reporter Analysis Settings for the SureSelect Cancer CGP DNA Assay

Field or menu Value(s) Usage guidelines

SNV/Indel Tumor-normal Use to perform SNV and indel calling with a matched or unmatched reference
Analysis mode sample. If no matched or unmatched reference sample is available for the analysis,
then the Analysis mode is set to Tumor-only and cannot be changed.

Tumor-only Use to perform SNV and indel calling without a reference sample.

SNV/Indel MBC (requires Select MBC deduplication mode, then select Hybrid consensus mode for the
Deduplication mode selection of Hybrid, deduplication settings recommended for high-sensitivity Cancer CGP DNA Assay
Single, or Duplex SNV/Indel analysis. Using these settings, reads that share the same MBC, genomic
consensus mode) position, library ID, and orientation are identified as duplicates, with consensus read
determination methods optimal for this assay at the recommended sequencing
depth. For any consensus mode selection, retaining the default Minimum number of
read pairs per MBC value(s) is recommended.

Positional Deduplication method based on read genomic position, library ID, and orientation
without consideration of MBC sequence. If preferred, this mode can be used for
Cancer CGP DNA Assay analysis.

SNV/Indel analysis Retain defaults for The Minimum variant allele frequency setting (with default value 0.05) may be
(multiple settings) recommended reduced to include variants at lower abundance in the sample or increased to
values exclude lower abundance variants. The associated Minimum reads supporting
variant allele setting may require co-adjustment. Somatic SNV/indel calling is only
done at genomic positions with coverage of at least 6 reads and for variants having
an alternative allele frequency of at least 0.001.

CNV analysis Retain defaults for Settings may be adjusted to include or exclude variants detected at different quality
(multiple settings) recommended scores or lengths. Alissa Reporter only considers a variant to be a putative CNV if it
values is at least 500 bp in length.

Translocation Retain defaults for The Minimum variant allele frequency setting (with default value 0.01) may be
analysis (multiple recommended reduced to include variants at lower abundance in the sample or increased to
settings) values exclude lower abundance variants. The associated Minimum reads supporting
variant allele setting may require co-adjustment. Only translocation variants having
an alternative allele frequency of at least 0.001 and read coverage of at least 1 are
reported.

TMB analysis Yes (default) or No Indicates if calculation of TMB (tumor mutational burden) is to be included in the
sample analysis. TMB analysis is not compatible with the use of an in silico filter.

MSI analysis Yes (default) or No Indicates if calculation of MSI (microsatellite instability) is to be included in the
sample analysis.

SureSelect Cancer CGP Assay User Guide 55

Table 43 Alissa Reporter Analysis Settings for the SureSelect Cancer CGP RNA Assay

Field or menu Value(s) Usage guidelines

RNA fusion analysis Positional Use of positional deduplication is generally recommended for Cancer CGP RNA
Deduplication mode samples.

None This option may be helpful for identifying low frequency variants in RNA samples
because more reads are retained for the variant calling analysis.

RNA fusion analysis Retain default Minimum total supporting reads setting of 2 is optimal for most RNA fusion
events. The total supporting reads is the sum of all junction reads and reads
from fusion-spanning read pairs.

Exon skipping analysis Retain default Alissa Reporter uses a Transcripts Per Kilobase Million (TPM) normalization
method for RNA abundance reporting, with separate TPM values reported for
the variant transcript (with exon skipping) and the normal transcript (without
exon skipping) in the sample. The minimum TPM ratio variant/variant+normal
setting controls the relative abundance of an exon skipping variant in the sample
required for a positive call. The default threshold value is 0.5. Agilent
recommends using known positive and negative samples for MET exon
14-skipping and EGFRvIII to verify and optimize this threshold setting. Calls
passing the threshold should be further evaluated for variant TPM value. A
minimum variant TPM of 10 is recommended to indicate sufficient expression
of the variant transcript for calling.

3 Once all run and sample attributes (required) and analysis settings (optional) are entered, click
the Upload files button.
To begin analysis immediately after the FASTQ files are uploaded, select Start analysis when
upload is completed checkbox. When selected, all analysis settings must be entered before file
upload begins.
4 If analysis was not initiated during file upload, analyze the run or individual samples after file
upload is complete. To analyze the full run, open the run from the List of Runs page, then click
Analyze run. To analyze an individual sample, open the sample from the List of Samples page,
then click Analyze sample. See the considerations for analysis settings in Table 42 (for DNA
assay) or Table 43 (for RNA assay) above.
5 Once analysis is complete, the Alissa Reporter software provides a variety of options for
viewing and reporting of sample data and run data including the options below:
• To view summary information for a sample or for a run click the icon for the sample or run
of interest then open the Overview tab.
• To generate a report in .pdf format that includes the full set of run and/or sample attributes,
QC metrics and a summary of analysis results, open the run page or the sample page then
from the Overview tab click the Create report button.
• To view the QC metrics results for the run or a specific sample, click the icon for the sample
or run of interest, then open the QC dashboard tab.
• To view the analysis results for a sample, click the icon for the sample of interest, then open
the tab for the relevant type of analysis (SNV/Indel, CNV, Translocation, TMB or MSI tab for
DNA analysis and RNA fusion or Exon skipping tab for RNA analysis). To view detailed
information on an individual variant, click the variant on the list then click one of the
available options for viewing the variant details, such as Open in view or Show pileup. The
available results display options vary for different genomic/transcriptomic features.

SureSelect Cancer CGP Assay User Guide 56

 • To create downloadable output files for the run or a sample, click the Download file button
from a run page or a sample page Overview tab. The available output file types include a QC
metrics text file and a variety of application-specific output files such as the Inaccessible
Regions BED file and variant results summaries in tabular format or Variant Call Format
(VCF).
Review the retention policies for uploaded sequencer files, data files and analysis
N OTE results files by clicking the Help icon (?) then clicking Go to help overview in the
Help dialog footer and browsing to the Data retention policy topic.

Analysis Considerations

Guidelines for key SureSelect Cancer CGP assay analysis considerations are provided below, with
details for analysis using Agilent’s Alissa Reporter software included where appropriate. Consult
the documentation for your selected analysis software for complete information on analysis
algorithm-based requirements, thresholds, precautions and limitations. For Agilent’s Alissa
Reporter, a full list of variant calling precautions and limitations is available for each assay in the
pdf-formatted Analysis Report generated from the dashboard for each analyzed sample or run.
• SNV/Indel variant allele frequency: Limitations to detection of SNVs and Indels depend on the
coverage and sequencing depth. The Alissa Reporter Cancer CGP Assay uses a default
SNV/Indel minimum variant allele frequency (VAF) setting of 0.05. It is possible to adjust this
setting to 0.001. However, detection of SNV and Indel variants present at <5% frequency may
require analysis using more than 40M reads and lowering this setting could increase the
number of false-positive variant calls. Given the expected range of VAF values and the
threshold for variant filtering based on VAF, some true-positive variant calls may be discarded
due to low VAF.
• Sex chromosome SNV/Indel variants: For SNV/indel calling on the sex chromosomes in
tumor-normal mode, the sex of the samples is used in the analysis. It is important to use target
and reference samples that have the same sex.
• Reference DNA processing: When using Agilent’s Alissa Reporter, CNV analysis and
SNV/Indel analysis in tumor-normal mode both require sequence data from a reference
sample (either matched or unmatched). It is recommended to include a matched
(non-tumorous tissue) reference for tumor-normal paired analysis. For both matched
reference and unmatched reference, it is recommended to process and sequence the
reference sample in the same run as the target samples. Alissa Reporter software allows
analysis using pre-established unmatched reference sample data from a prior run. However,
the potential bias due to batch differences may increase the copy number noise and negatively
impact the accuracy of calling. Agilent’s OneSeq Human Reference DNA or DNA from any
sex-matched control sample that does not contain aberrations can be used as an unmatched
reference sample for the Cancer CGP DNA application.
• CNV analysis: CNV calls should be reviewed and verified with consideration of adherence to
the reference sample requirements and other variant calling factors.
� To enable CNV analysis, sequence data from a reference sample (either matched or unmatched)
without copy number aberrations in the regions of interest is required. In order for a reference sample
to be considered matched, it must be collected from the same source as the target sample, but from
an area consisting of normal/non-tumorous tissue. Unmatched reference samples may also be used.
For Agilent’s Alissa Reporter software, using a matched reference sample improves the sensitivity of
the CNV calling algorithm. Co-processing of reference samples and experimental target samples is
recommended.

SureSelect Cancer CGP Assay User Guide 57

 � CNV calling on the sex chromosomes depends on the sex of the sample in the analysis. It is important
to provide accurate sex information for each sample.
� Consult the selected analysis software documentation for any additional algorithm-based CNV calling
reference sample requirements, precautions and limitations.
• Targeted translocations: The SureSelect Cancer CGP DNA assay probe design targets
selected regions of oncogenic driver genes at specific reported translocation break-ends. A
translocation with neither mate read in the targeted regions will not be detected regardless of
the abundance of the translocation in the sample.
• Translocations and repetitive sequences: Translocations frequently occur in intronic and
intergenic regions that are more likely to contain repetitive sequences with lower capture
specificity than exonic regions. Due to the low complexity of these regions the sequencing
reads are difficult to align accurately to the reference genome. Misaligned reads can result in
spurious translocation event calls. Agilent’s Alissa Reporter software applies filters to
minimize these events.
• TMB analysis: The exonic genome coverage size (sum of exonic region sequence) of the
SureSelect Cancer CGP Assay Probe DNA is 1.605 Mb. Using Agilent’s Alissa Reporter
SureSelect Cancer CGP Assay analysis, TMB is calculated as the ratio of the number of
variants detected in a sample to the effective genome coverage in the run. TMB determination
is most accurate for samples with coverage ≥1.6 Mb. Runs with 1.0 to 1.6 Mb coverage may
generate over-estimated or under-estimated TMB values. Alissa Reporter software does not
make TMB determinations for runs with coverage <1 Mb.
• MSI analysis: The SureSelect Cancer CGP Assay Probe DNA targets 288 sites available for
microsatellite instability (MSI) determinations. Coverage for typical samples is in the range of
250-280 sites, and MSI determination is most accurate for samples having coverage in this
range. Samples with coverage in <150 of the available sites may generate over-estimated or
under-estimated MSI values. Using Agilent’s Alissa Reporter SureSelect Cancer CGP Assay
analysis, MSI determinations are not made for samples with coverage of <150 sites.
• RNA fusions: The SureSelect Cancer CGP RNA Assay probe design enables detection of RNA
fusions in 80 genes, regardless of partner. Agilent’s Alissa Reporter software identifies
sequencing reads that correspond to both the targeted transcript and a partner transcript
(which may or may not also be targeted in the assay). It is possible for these reads to occur in
the absence of a fusion event, for example read-through transcription of neighboring genes or
through library preparation or sequencing artifacts. Results should be interpreted by trained
personnel.
• RNA exon-skipping: The SureSelect Cancer CGP RNA assay probe design targets two specific
splice variants, EGFRvIII and MET Exon14-skipping. In normal samples, a low-level of variant
transcripts may be detected. To minimize false positive calls, use known positive and negative
samples for a specific exon skipping locus of interest to define the appropriate threshold for
calling the exon skipping event in the selected analysis software. Calls passing the threshold
should be further evaluated for variant transcripts per million (TPM) value. A minimum variant
TPM of 10 is recommended to indicate sufficient expression of the variant transcript for
calling.

SureSelect Cancer CGP Assay User Guide 58

 Agilent SureSelect Cancer CGP Assay
User Guide

6
Appendix 1: SureSelect Cancer CGP Automation
Automation Overview 59
Magnis Automation Workflow 60
Bravo Automation Workflow 61

Automation Overview

NGS library preparation for the SureSelect Cancer CGP Assay can be automated using the
solutions detailed in Table 44. Review the workflow outline for your automation system on
page 60 for the Magnis system or on page 61 for the Bravo system. These sections include links
to the relevant SureSelect XT HS2 automation user guides and important tips for optimizing each
automation protocol for the SureSelect Cancer CGP Assay.

Table 44 Ordering information for SureSelect Cancer CGP Assay Automation Solutions

Description Agilent Reagent Modules Included

Part
Probe DNA Library Prep + RNA Library Prep + Capture Beads, Enzymatic DNA
Number
Hyb Reagents Hyb Reagents Purification Fragmentation
Beads

Magnis SureSelect Cancer CGP Assay Kits (32 Samples or 96 Samples)

Magnis Cancer CGP DNA G9777A DNA  ×  
Reagent Kit, 32 Reactions (Index 1-32) (not applicable)

Magnis Cancer CGP DNA G9777B DNA  ×  

Reagent Kit, 96 Reactions (not applicable)
(Index 1-96 OR 97-192)

Magnis Cancer CGP RNA G9777C RNA ×   ×

Reagent Kit, 32 Reactions (not applicable) (not applicable)
(Index 1-32)

Magnis Cancer CGP RNA G9777D RNA ×   ×

Reagent Kit, 96 Reactions (not applicable) (Index 1-96 OR 97-192) (not applicable)

Agilent Bravo Automation SureSelect Cancer CGP Assay Kits (96 Samples)
SureSelect Cancer CGP G9966B DNA    —
Assay DNA & RNA Kit, & RNA† (Index 1-96) (Index 97-192) (optional; order
96 Samples Each, Auto p/n 5191-4080)

SureSelect Cancer CGP G9967C DNA  ×  —

Assay DNA Kit, 96 Samples, (not applicable) (optional; order
(Index 1-96)
Auto p/n 5191-4080)

SureSelect Cancer CGP G9968C RNA ×   ×

Assay RNA Kit, 96 Samples, (not applicable) (Index 97-192) (not applicable)
Auto

59
Magnis Automation Workflow

Pre-run instrument and labware preparation

Before you begin, review the appropriate user guide(s) below to familiarize yourself with the
automation workflow. Consult the Materials Required section and ensure that all materials needed
for automated NGS library preparation are available in your laboratory. Verify that your Magnis
instrument is equipped with the necessary run protocols and firmware.

Table 45 Magnis automation parameters

SureSelect Cancer Magnis Automation Magnis Protocol used for Magnis Instrument
CGP Assay User Guide Link NGS Library Preparation Firmware Version Required
DNA G9751-90000 SSEL-DNA-XTHS2-ILM v1.3 or later
RNA G9752-90000 SSEL-RNA-XTHS2-ILM v1.4 or later

Pre-run sample preparation

Prepare and qualify the DNA or RNA samples as directed in the appropriate Magnis SureSelect XT
HS2 user guide. All samples for Magnis-automated SureSelect Cancer CGP assays must be
prepared in 1X Low TE Buffer (10 mM Tris-HCl, pH 7.5-8.0, 0.1 mM EDTA), using the volumes
specified in Table 46.

Table 46 Magnis sample input parameters

Nucleic Acid Input Type Magnis Protocol used for Input Amount Options* Sample Volume Required
NGS Library Preparation for Automation Protocol
Unsheared DNA (from SSEL-DNA-XTHS2-ILM 10 ng, 50 ng, 100 ng, or 200 ng 14 µL
high-quality or FFPE samples) with enzymatic
fragmentation
Covaris-sheared DNA (from SSEL-DNA-XTHS2-ILM 10 ng, 50 ng, 100 ng, or 200 ng 50 µL
high-quality or FFPE samples) without enzymatic
fragmentation
Intact RNA or good-quality FFPE SSEL-RNA-XTHS2-ILM 10 ng, 50 ng, 100 ng, or 200 ng 10 µL
RNA samples
Poor-quality FFPE RNA samples SSEL-RNA-XTHS2-ILM 50 ng, 100 ng, or 200 ng 10 µL

* Input amounts listed in this table include all options available in the Magnis software for each specific Magnis protocol and input type. The
SureSelect Cancer CGP assay is optimized for sample input amounts of 50 ng DNA or RNA. For lower-quality FFPE samples, assay performance
may be improved by increasing the amount of DNA or RNA input to 100 ng or 200 ng. Use of 10 ng input DNA or RNA is supported for some
Magnis run types, but may lead to reduced performance for the SureSelect Cancer CGP assay.

For optimal SureSelect Cancer CGP assay performance, use of 50 ng input is recommended for
most samples. For low-quality FFPE samples, assay performance may be improved by increasing
the amount of DNA or RNA input to 100 ng or 200 ng. Some Magnis run types also allow use of
10 ng DNA or RNA input, however use of input <50 ng for the SureSelect Cancer CGP Assay may
lead to lower target coverage and reduced detection of low-frequency variants.

SureSelect Cancer CGP Assay User Guide 60

 All experimental samples processed in the same eight (8)-sample Magnis run should be of the
same input amount and same input type.
DNA assay runs analyzing FFPE samples can include a high-quality DNA control
N OTE sample in the same run; select the Sample Type option of FFPE DNA during run
setup for these runs.
For RNA assay runs analyzing FFPE samples, any control intact RNA samples
must be processed in a separate Magnis run with Intact RNA selected as Sample
Type during run setup. Intact RNA samples are not properly fragmented under
FFPE RNA run conditions.

Performing the Magnis NGS library preparation run

Perform automated NGS library preparation as directed in the appropriate Magnis user guide,
through the final library quantification step.

Post-run library processing

Follow the post-run library processing guidelines provided in this publication, starting with pooling
the quantified NGS libraries for multiplexed sequencing, as outlined on page 49. Use the NGS
support resources in this publication for the Magnis-processed libraries (page 50 to page 57 and
index sequences provided on page 79 to page 83).

Bravo Automation Workflow

Before you begin, review the appropriate user guide(s) below to familiarize yourself with the
automation workflows. Consult the Materials Required section and ensure that all materials
needed for Bravo-automated NGS library preparation are available in your laboratory. Verify that
your Agilent NGS Workstation or Bravo instrument is equipped with the required VWorks software
forms.

Table 47 Bravo automation summary

SureSelect Cancer CGP Assay Bravo Automation User Guide Link VWorks Software Form
NGS Workstation (Option B)* NGS Bravo (Option A)
DNA G9985-90010 G9985-90020 SureSelect XT HS2 DNA Form
RNA G9993-90010 G9993-90020 SureSelect XT HS2 RNA Form

* The Bravo NGS Workstation Option B+, with an on-deck thermal cycler, is also available with protocols for processing SureSelect XT HS2 DNA
assays. See publication G9985-90015 for protocol details. The provided SureSelect XT HS2 DNA assay protocols can be used for processing
SureSelect Cancer CGP Assay samples, after verification of the required performance in your laboratory (see Table 50 on page 65 for PCR
cycle numbers and other run attributes). At the time of this publication, SureSelect XT HS2 RNA assay automation protocols are not available
for the Bravo NGS Workstation Option B+.

SureSelect Cancer CGP Assay User Guide 61

 Pre-run sample preparation and qualification
Prepare, qualify, and quantify total RNA and gDNA samples according to the instructions provided
in this publication (see Table 48 for links to the appropriate sections). For workflows that include
mechanical DNA shearing, also use the instructions provided on page 27 of this publication for
the fragmentation workflow segment.
Samples must be placed in the appropriate Bravo-compatible 96-well plate for further processing,
using the volumes and solvents specified in Table 48. For optimal SureSelect Cancer CGP assay
performance, use of 50 ng input is recommended for most samples. For low-quality FFPE
samples, assay performance may be improved by increasing the amount of DNA or RNA input to
up to 200 ng. Runs may be set up using as little as 10 ng DNA or RNA input, with possible negative
impacts on target coverage and detection of low-frequency variants.

Table 48 Bravo automation sample input parameters

SureSelect Cancer Fragmentation Method Link to Sample Sample Composition Required

CGP Assay Prep/QC Steps to Begin Automation
RNA Bravo-automated chemical fragmentation page 17 to page 18 10 to 200 ng* total RNA in 10 µL
(only high-quality intact RNA samples nuclease-free water (starting
require fragmentation; see Table 49 on sample for VWorks protocol
page 63) Fragmentation_XT_HS2_RNA)
DNA Bravo-automated enzymatic fragmentation page 24 to page 25 10 to 200 ng* gDNA in 15 µL
(see Table 50 on page 65) nuclease-free water (starting
sample for VWorks protocol
EnzFrag_XT_HS2_ILM)
Mechanical shearing (non-automated) page 24 to page 25 and 10 to 200 ng* DNA fragments in
page 27 to page 28 50 µL 1X Low TE Buffer (starting
sample for VWorks runset
LibraryPrep_XT_HS2_ILM)

* Follow the input amount recommendations provided in this publication (use ≥50 ng DNA or RNA input, based on sample quality, for optimal
performance). Runs may be set up using as little as 10 ng DNA or RNA input for automated processing using SureSelect XT HS2 chemistry,
with possible negative impacts on target coverage and detection of low-frequency variants.

All experimental samples processed in the same plate should be of the same input type and input
amount to allow amplification and fragmentation, where applicable, under the same conditions.
RNA assay runs analyzing FFPE samples can include high-quality RNA control
N OTE samples on the same plate, with the run modifications listed on page 64.
DNA assay runs analyzing FFPE samples can include high-quality DNA control
samples on the same plate, with the run modifications listed on page 66.

Performing the NGS library preparation run

For a summary of the VWorks protocols used for SureSelect Cancer CGP automation, see
Table 49 on page 63 for the RNA Assay and see Table 50 on page 65 for the DNA Assay.
To set up and run each VWorks automation protocol, use the detailed instructions provided in the
SureSelect XT HS2 automation user guides listed in Table 47 on page 61.
RNA and DNA samples are processed separately through the Bravo automation protocols, using
VWorks protocols initiated from the VWorks Form corresponding to the DNA or RNA sample type.

SureSelect Cancer CGP Assay User Guide 62

 RNA Assay automation protocols
Table 49 RNA Assay--overview of VWorks automation protocols and runsets

Workflow Step Substep VWorks Protocols* Used for Agilent Notes

NGS Workstation Automation
AMPure XP For use in Second-Strand Synthesis AMPureXP_Aliquot (Case
Bead Aliquoting runset Second-Strand) You can prepare all AMPure XP
For use in Library Prep runset AMPureXP_Aliquot (Case Library bead plates needed for
Prep) same-day use at the start of the
day’s workflow to reduce
For use in Pre-Capture PCR AMPureXP_Aliquot (Case delays between steps. Keep
purification protocol Pre-Capture PCR) prepared plates at 4°C for up to
For use in Post-Capture PCR AMPureXP_Aliquot (Case 24 hours.
purification protocol Post-Capture PCR)
RNA Mix RNA samples with the 2X Fragmentation_XT_HS2_RNA Process intact RNA and FFPE
Preparation and Priming Buffer RNA assays on separate
cDNA plates. Do the 94°C
Synthesize first-strand cDNA FirstStrandcDNA_XT_HS2_RNA
Conversion fragmentation step only for
Synthesize and purify second-strand SecondStrand_XT_HS2_RNA intact RNA samples. If intact
cDNA control included on FFPE
sample plate, see page 64.
Prepare and purify Runset LibraryPrep_XT_HS2_ILM
—
molecular-barcoded DNA libraries
Amplify DNA libraries with dual Pre-CapPCR_XT_HS2_ILM Use 12 PCR cycles for
indexing primer pairs high-quality RNA input or
15 PCR cycles for FFPE RNA
Library input. If intact RNA control was
Preparation included in FFPE sample run,
see page 64.
Purify indexed DNA libraries using AMPureXP_XT_HS2_ILM (Case Use instructions for single-plex
AMPure XP beads Pre-Capture PCR – SinglePlex) hyb/post-capture pooling
Set up plates for library QC using TS_D1000 May also be performed
Agilent TapeStation platform manually (see page 37)
Aliquot 200 ng of prepped cDNA Aliquot_Libraries Use instructions for single-plex
libraries into hybridization plate hyb; may also be performed
manually (see page 40)
Hybridization
and Capture Hybridize prepped libraries (target Hyb_XT_HS2_ILM Use instructions for probes
enrichment) ≥3 Mb, single probe in all rows
Capture and wash DNA hybrids Runset SSELCapture&Wash_XT_HS2 —
Post-Capture Amplify target-enriched libraries Post-CapPCR_XT_HS2_ILM Use 13 PCR cycles
Sample
Purify enriched, amplified libraries AMPureXP_XT_HS2_ILM (Case
Processing —
using AMPure XP beads Post-Capture PCR)
Set up plates for final library QC TS_HighSensitivity_D1000 May also be performed
using Agilent TapeStation platform manually (see page 46)

* Use the SureSelect XT HS2 RNA VWorks form to open each automation protocol. Some of the protocols and runsets accessed from the Sure-
Select XT HS2 DNA Form are not compatible with the reagent and labware positioning specifications used for the RNA assay.

SureSelect Cancer CGP Assay User Guide 63

 Running intact RNA controls with FFPE RNA samples
• Perform the Fragmentation_XT_HS2_RNA protocol liquid-handling steps as directed in the
Bravo automation user guide, starting with all samples on the same plate. Once complete,
remove the plate from the Bravo deck to ice, then transfer the intact control RNA sample(s) to
well(s) of a fresh strip tube. Perform the 94°C thermal cycler incubation step described in the
Bravo automation user guide using only the intact control RNA strip. Once complete, transfer
each fragmented control sample back to its original FFPE RNA sample plate well for further
processing.
• Perform the Pre-CapPCR_XT_HS2_ILM protocol liquid-handling steps as directed in the Bravo
automation user guide, starting with all samples on the same plate. Once complete, remove
the plate from the Bravo deck to ice, then transfer the control RNA library amplification
reaction(s) to well(s) of a fresh strip tube. Amplify the FFPE and control sample libraries using
separate thermal cyclers using the amplification cycle number appropriate for each sample
type. Once the thermal cycling programs are complete, transfer each control library from the
control strip back to its original FFPE RNA sample plate well for further processing.

SureSelect Cancer CGP Assay User Guide 64

 DNA Assay automation protocols
Table 50 DNA Assay--overview of VWorks automation protocols and runsets

Workflow step Substep VWorks Protocols* Used for Agilent Notes

NGS Workstation Automation
For use in Library Prep runset AMPureXP_Aliquot (Case Library You can prepare all AMPure XP
Prep) bead plates needed for
same-day use at the start of
AMPure XP For use in Pre-Capture PCR AMPureXP_Aliquot (Case
the day’s workflow to reduce
Bead Aliquoting purification protocol Pre-Capture PCR)
delays between steps. Keep
For use in Post-Capture PCR AMPureXP_Aliquot (Case prepared plates at 4°C for up to
purification protocol Post-Capture PCR) 24 hours.
Shear DNA samples using EnzFrag_XT_HS2_ILM Process intact DNA and FFPE
enzymatic fragmentation DNA assays on separate
plates. If intact control included
Dilute fragmented samples to EnzFrag_Dil_XT_HS2_ILM
in FFPE DNA assay, see
appropriate concentration
Enzymatic DNA page 66.
Fragmentation Enzymatic DNA fragmentation
may be replaced with Covaris
DNA shearing using manual
liquid handing and shearing
steps (see page 27).
Prepare and purify Runset LibraryPrep_XT_HS2_ILM
—
molecular-barcoded DNA libraries
Amplify DNA libraries with dual Pre-CapPCR_XT_HS2_ILM Use 9 PCR cycles for
indexing primer pairs high-quality DNA input or 12
PCR cycles for FFPE DNA
Library input. If intact DNA control was
Preparation included in FFPE sample run,
see page 66.
Purify indexed DNA libraries using AMPureXP_XT_HS2_ILM (Case Use instructions for single-plex
AMPure XP beads Pre-Capture PCR – SinglePlex) hyb/post-capture pooling
Set up plates for library QC using TS_D1000 May also be performed
Agilent TapeStation platform manually (see page 37)
Aliquot 500-1000 ng of prepped Aliquot_Libraries Use instructions for single-plex
libraries into hybridization plate hyb; may also be performed
manually (see page 40)
Hybridization
and Capture Hybridize prepped libraries (target Hyb_XT_HS2_ILM Use instructions for probes
enrichment) ≥3 Mb, single probe in all rows
Capture and wash DNA hybrids Runset SSELCapture&Wash_XT_HS2 —
Post-Capture Amplify target-enriched libraries Post-CapPCR_XT_HS2_ILM Use 13 PCR cycles
Sample
Purify enriched, amplified libraries AMPureXP_XT_HS2_ILM (Case
Processing —
using AMPure XP beads Post-Capture PCR)
Set up plates for final library QC TS_HighSensitivity_D1000 May also be performed
using Agilent TapeStation platform manually (see page 46)

* Use the SureSelect XT HS2 DNA VWorks form to open each automation protocol. Some of the protocols and runsets accessed from the Sure-
Select XT HS2 RNA Form are not compatible with the reagent and labware positioning specifications required for the DNA assay.

SureSelect Cancer CGP Assay User Guide 65

 Running intact DNA controls with FFPE DNA samples
• For workflows including enzymatic fragmentation, perform the EnzFrag_XT_HS2_ILM protocol
liquid-handling steps as directed in the Bravo automation user guide with all samples on the
same plate. Once complete, all samples can be fragmented together, using the thermal cycler
program for FFPE DNA samples provided in the Bravo automation user guide. After
fragmentation, resume the workflow for the sample plate with the EnzFrag_Dil_XT_HS2_ILM
protocol.
• For workflows including mechanical shearing, use the shearing conditions specified for each
sample type in this publication (see page 27 to page 28). Once complete, all sheared DNA
samples can be placed on the same plate for further automated processing, starting with the
LibraryPrep_XT_HS2_ILM runset.
• Perform the Pre-CapPCR_XT_HS2_ILM protocol liquid-handling steps as directed in the Bravo
automation user guide, starting with all samples on the same plate. Once complete, remove
the plate from the Bravo deck to ice, then transfer the control DNA library amplification
reaction(s) to well(s) of a fresh strip tube. Amplify the FFPE and control sample libraries on
separate thermal cyclers using the amplification cycle number appropriate for each sample
type. Once the thermal cycling programs are complete, transfer each control library from the
control strip back to its original FFPE DNA sample plate well for further processing.

Post-run library processing

Follow the post-run library processing guidelines provided in this publication, starting with pooling
the quantified NGS libraries for multiplexed sequencing, as outlined on page 49. Use the NGS
support resources in this publication for the Magnis-processed libraries (page 50 to page 57 and
index sequences provided on page 79 to page 83).

SureSelect Cancer CGP Assay User Guide 66

 Agilent SureSelect Cancer CGP Assay
User Guide

7
Appendix 2: SureSelect Cancer Tumor-Specific
Assays
Overview of SureSelect Cancer Tumor-Specific Assays 67
Materials Required for SureSelect Cancer Tumor-Specific Assays 67
Running the SureSelect Cancer Tumor-Specific Assay 69

Overview of SureSelect Cancer Tumor-Specific Assays

SureSelect Cancer Tumor-Specific Assays have been developed for several specific solid tumor
types, in order to interrogate a variety of genomic features including SNVs, Indels, CNVs and
translocations at key loci for each tumor type. See the Cancer NGS Assays page at Agilent.com
for the most current list of tumor-specific assays available and for additional details on the
genomic features interrogated by each of the SureSelect Cancer Assay Probes.
Running the SureSelect Cancer Assay requires separate purchase of the relevant SureSelect
Cancer Assay Probe and a SureSelect XT HS2 DNA Reagent Kit. Target-enriched NGS libraries are
prepared from gDNA samples using this set of components using the protocols provided in this
publication with the minor modifications detailed in this Appendix. Once sequencing data is
collected for the assay samples, analysis is performed using the appropriate NGS analysis
software tool(s) for the variant discovery goals of your research.

Materials Required for SureSelect Cancer Tumor-Specific Assays

Running the SureSelect Cancer Assay requires the components listed below:
• SureSelect Cancer Assay DNA Probe (see Table 51 for a list of probes available at the time of
this publication)
• SureSelect XT HS2 DNA Reagent Kit with Fast-Hyb, Post-capture pooling target enrichment
reagents (seeTable 52 on page 68 for a list of compatible reagent kits)
• Additional reagents and equipment required for DNA assays using the selected sample type
detailed in Table 2 on page 12 through Table 4 on page 14

Table 51 SureSelect Cancer Assay Probes--Select One

Assay Probe Design ID Agilent Part Number

16 Hybs 96 Hybs*
SureSelect Cancer Lung Assay Probe, DNA A3464871 p/n 5282-0060 p/n 5282-0061
SureSelect Cancer Colon Assay Probe, DNA A3464881 p/n 5282-0062 p/n 5282-0063
SureSelect Cancer Pancreas Assay Probe, DNA A3464891 p/n 5282-0064 p/n 5282-0065
SureSelect Cancer Kidney Assay Probe, DNA A3464901 p/n 5282-0066 p/n 5282-0067
SureSelect Cancer Bladder Assay Probe, DNA A3464911 p/n 5282-0068 p/n 5282-0069

* Compatible with both manual-processing and Bravo-automated processing of 96 samples.

67
 .

Table 52 Ordering Information for Compatible SureSelect XT HS2 DNA Kits

Agilent Part Samples Library Prep Kit Target Enrichment Kit Capture Beads, Enzymatic DNA
Number Processed (Index Pairs Included) Purification Beads Fragmentation Kit
Complete SureSelect XT HS2 DNA Starter Kit (16 Samples)
G9982A 16*    
Index 1–16 (Fast-Hyb, Post-Cap Pool)

SureSelect XT HS2 DNA Reagent Kits with AMPure® XP† & Streptavidin Beads (96 Samples)
G9984A 96‡    Optional
(order Agilent
Index 1–96 (Fast-Hyb, Post-Cap Pool)
p/n 5191-4080 for
‡
G9984B 96  manual processing
or p/n 5191-6764 for
Index 97–192 Bravo-automation)
G9984C 96‡ 
Index 193–288

G9984D 96‡ 
Index 289–384

SureSelect XT HS2 DNA Reagent Kits

G9981A 16*   × Optional
(order Agilent
Index 1–16 (Fast-Hyb, Post-Cap Pool) Requires separate purchase
p/n 5191-4080 for
of materials below:
G9983A 96‡  manual processing
• Dynabeads MyOne
or p/n 5191-6764 for
Index 1–96 Streptavidin T1 from
Bravo-automation)
Thermo Fisher Scientific
G9983B 96‡  • AMPure® XP Kit from
Index 97–192 Beckman Coulter

G9983C 96‡ 
Index 193–288

G9983D 96‡ 
Index 289–384

* Kits are compatible with manual-processing of 16 samples using the protocols and supported run sizes described in this publication.
† AMPure, Beckman, and Beckman Coulter are trademarks or registered trademarks of Beckman Coulter, Inc.
‡ Kits are compatible with manual-processing of 96 samples using the protocols and supported run sizes described in this publication. Kits are
also compatible with Bravo-automated processing of 96 samples using the supported run configurations described in the Bravo automation
user guides (see Table 47 on page 61).

SureSelect Cancer CGP Assay User Guide 68

Running the SureSelect Cancer Tumor-Specific Assay

• Consult “Sample requirements” on page 9 before you begin.

• Prepare, qualify, and fragment gDNA samples as directed on page 24 to page 28.
• Prepare SureSelect XT HS2 libraries as directed on page 30 to page 37, using the instructions
specific for DNA samples.
• Target-enrich the libraries using the appropriate SureSelect Cancer Probe. Follow the
instructions provided on page 39 to page 46, with the following modification. In the
post-capture PCR thermal cycler program in Table 33 on page 43, amplify using 16 PCR cycles
(replacing 13 cycles) in segment 2.
• Process the target-enriched libraries for NGS as described on page 48 to page 52, with the
following modification. As a replacement for the sequencing depth recommendations
provided on page 48, Agilent recommends using ≥5M reads per sample for SureSelect Cancer
Colon, Kidney, and Bladder Assays or ≥7.5M reads per sample for SureSelect Cancer Lung and
Pancreas Assays.
• The SureSelect Cancer Assay probes are compatible with processing using Agilent’s Magnis
and Bravo automation systems as detailed below. Performance specifications have not,
however, been specifically verified for libraries enriched using these automation systems.
• Magnis automation is available for the SureSelect Cancer Assay probe designs when the
appropriate SureSelect Cancer Assay Probe Design ID (see Table 51 on page 67) is used to
order a custom Magnis SureSelect XT HS2 DNA Reagent Kit. Visit Agilent’s SureDesign site for
custom Magnis probe design and kit ordering information. See page 60 to page 61 for
additional Magnis automation guidelines. Alternatively, Magnis automation can be completed
using the probe products listed in Table 51 on page 67 in conjunction with Magnis SureSelect
XT HS2 DNA (No Probe) Reagent Kits (Agilent P/N G9750B), provided with empty probe input
strips which can be filled with the appropriate SureSelect Cancer Assay probe(s). See the
Magnis SureSelect XT HS2 DNA user guide for more information on use of self-filled probe
strips with the Magnis automation protocol SSEL-DNA-XTHS2-EPIS-ILM.
• For Bravo automation of the SureSelect Cancer Assays, use the instructions provided on
page 61 to page 62 and page 65 to page 66 with the following modification. When running the
Post-CapPCR_XT_HS2_ILM Bravo protocol listed in Table 50 on page 65, use 16 PCR cycles
(replacing 13 cycles). The 96-Hyb SureSelect Cancer Assay Probes listed in Table 51 on
page 67, along with the 96-Sample SureSelect XT HS2 DNA Reagent Kits listed in Table 52 on
page 68 are compatible with Bravo-automated processing.
• For co-analysis of RNA transcriptome variants in samples analyzed using a SureSelect Cancer
assay, Agilent recommends analyzing the samples using the SureSelect Cancer CGP RNA
probe in parallel with the selected SureSelect Cancer Assay DNA probe. See page 8 for a
description of the variants targeted by the SureSelect Cancer CGP RNA Assay and see
Table 59 on page 77 for CGP RNA probe ordering information.

SureSelect Cancer CGP Assay User Guide 69

 Agilent SureSelect Cancer CGP Assay
User Guide

8
Appendix 3: SureSelect Cancer Custom Panel
Assays
Overview of SureSelect Cancer Custom Panel Assays 70
Materials Required for SureSelect Cancer Custom Panel Assays 70
Running a SureSelect Cancer Custom Panel Assay 72

Overview of SureSelect Cancer Custom Panel Assays

SureSelect Cancer Custom Panels can include content from the pre-defined SureSelect Cancer
CGP DNA Assay panel or SureSelect Cancer Tumor-Specific Assay panels, along with
user-defined content. The dedicated design tool in SureDesign provides a seamless
customization process for including or excluding specific targets in the existing panels along with
adding content not found in existing panels. To begin the SureSelect Cancer Custom Panel design
process, visit Agilent’s SureDesign site.
Running the assay requires separate purchase of a SureSelect Cancer Custom Panel and a
SureSelect XT HS2 DNA Reagent Kit. Target-enriched NGS libraries are prepared from gDNA
samples with this set of components using the protocols provided in this publication with the
minor modifications detailed in this Appendix. Once sequencing data is collected for the assay
samples, analysis is performed using the appropriate NGS analysis software tool(s) for the variant
discovery goals of your research.

Materials Required for SureSelect Cancer Custom Panel Assays

Running the assay requires the components listed below:
• SureSelect Cancer Custom Panel probe. See Table 53 for a list of user-customized probes
available from SureDesign at the time of this publication. The SureSelect Cancer Custom Panel
probes are also available for Magnis automation; see page 72 for more information.
• SureSelect XT HS2 DNA Reagent Kit with Fast-Hyb, Post-capture pooling target enrichment
reagents (seeTable 54 on page 71 for a list of compatible reagent kits)
• Additional reagents and equipment required for DNA assays using the selected sample type
detailed in Table 2 on page 12 through Table 4 on page 14

Table 53 SureSelect Cancer Custom Panel options

Custom Design Type Agilent Part Number

16 Hybs 96 Hybs 96 Hybs Auto*
SureSelect Cancer CGP Catalog + Tier 1 Custom p/n 5282-0170 p/n 5282-0171 p/n 5282-0172
SureSelect Cancer Custom Tier 1 p/n 5282-0164 p/n 5282-0165 p/n 5282-0166
SureSelect Cancer Custom Tier 2 p/n 5282-0167 p/n 5282-0168 p/n 5282-0169

* Compatible with Bravo-automated processing of 96 samples.

70
Table 54 Ordering Information for Compatible SureSelect XT HS2 DNA Kits

Agilent Part Samples Library Prep Kit Target Enrichment Kit Capture Beads, Enzymatic DNA
Number Processed (Index Pairs Included) Purification Beads Fragmentation Kit
Complete SureSelect XT HS2 DNA Starter Kit (16 Samples)
G9982A 16*    
Index 1–16 (Fast-Hyb, Post-Cap Pool)

SureSelect XT HS2 DNA Reagent Kits with AMPure® XP† & Streptavidin Beads (96 Samples)
G9984A 96‡    Optional
Index 1–96 (Fast-Hyb, Post-Cap Pool) (order Agilent
p/n 5191-4080 for
G9984B 96‡  manual processing
or p/n 5191-6764 for
Index 97–192 Bravo-automation)
G9984C 96‡ 
Index 193–288

G9984D 96‡ 
Index 289–384
SureSelect XT HS2 DNA Reagent Kits
G9981A 16*   × Optional
(order Agilent
Index 1–16 (Fast-Hyb, Post-Cap Pool) Requires separate purchase
p/n 5191-4080 for
of materials below:
G9983A 96‡  manual processing
• Dynabeads MyOne
or p/n 5191-6764 for
Index 1–96 Streptavidin T1 from
Bravo-automation)
Thermo Fisher Scientific
G9983B 96‡  • AMPure® XP Kit from
Index 97–192 Beckman Coulter

G9983C 96‡ 
Index 193–288
‡
G9983D 96 
Index 289–384

* Kits are compatible with manual-processing of 16 samples using the protocols and supported run sizes described in this publication.
† AMPure, Beckman, and Beckman Coulter are trademarks or registered trademarks of Beckman Coulter, Inc.
‡ Kits are compatible with manual-processing of 96 samples using the protocols and supported run sizes described in this publication. Kits are
also compatible with Bravo-automated processing of 96 samples using the supported run configurations described in the Bravo automation
user guides (see Table 47 on page 61).

SureSelect Cancer CGP Assay User Guide 71

Running a SureSelect Cancer Custom Panel Assay

Prepare NGS libraries from gDNA samples using the protocols provided in this publication with
the minor modifications detailed here.
• Consult “Sample requirements” on page 9 before you begin.
• Prepare, qualify, and fragment gDNA samples as directed on page 24 to page 28.
• Prepare SureSelect XT HS2 libraries as directed on page 30 to page 37, using the instructions
specific for DNA samples.
• Target-enrich the libraries using the SureSelect Cancer Custom Panel probe. Follow the
instructions provided on page 39 to page 46, with the following modification. In the
post-capture PCR thermal cycler program in Table 33 on page 43 (Segment 2), amplify using
the PCR cycle number appropriate for the custom design size (see Table 55 for guidelines).
Cycle numbers in Table 55 are estimates to give yield of 10–20 nM; adjustment may be
necessary based on the specific probe design and sample type/quality used in the assay.

Table 55 Post-capture PCR cycle number recommendations

Design Size Post-capture PCR Cycles

Probes <500 kb 16 cycles

Probes 0.5–2 Mb 14 cycles

Probes 2–3 Mb 13 cycles

Probes 3–6 Mb 12 cycles

Probes >6 Mb 11 cycles

• Process the target-enriched libraries for NGS as described on page 48 to page 52, with the
following modification. As a replacement for the sequencing depth recommendations
provided on page 48, Agilent recommends targeting a raw sequencing depth of ≥3000X reads
per sample. The recommended minimum raw reads for a specific design size is calculated as
follows for paired-end sequencing:
Million reads = [(Mb design size*3000)/sequencing read length]
• SureSelect Cancer Custom Panel Assays are compatible with processing using Agilent’s
Magnis and Bravo automation systems as detailed below.
• Magnis automation is available for the SureSelect Cancer Custom Panel Assays. Contact your
local Agilent representative for assistance with ordering Magnis SureSelect XT HS2 DNA
Reagent Kits with SureSelect Cancer Custom Panel probe strips. Alternatively, Magnis
automation can be completed using the probe products listed in Table 53 on page 70 in
conjunction with Magnis SureSelect XT HS2 DNA (No Probe) Reagent Kits (Agilent P/N
G9750B), provided with empty probe input strips which can be filled with the appropriate
SureSelect Cancer Custom Panel probe(s). See the Magnis SureSelect XT HS2 DNA user guide
for more information on use of self-filled probe strips with the Magnis automation protocol
SSEL-DNA-XTHS2-EPIS-ILM. See page 60 to page 61 for additional Magnis automation
guidelines.

SureSelect Cancer CGP Assay User Guide 72

 • For Bravo automation of the SureSelect Cancer Custom Panel Assays, use the instructions
provided on page 61 to page 62 and page 65 to page 66 with the following modification. When
running the Post-CapPCR_XT_HS2_ILM Bravo protocol listed in Table 50 on page 65, amplify
using the PCR cycle number appropriate for the custom design size (see Table 55 on page 72
for guidelines). Cycle numbers in Table 55 are estimates to give yield of 10–20 nM; adjustment
may be necessary based on the specific probe design and sample type/quality used in the
assay. See Table 53 on page 70 for Bravo-automation compatible probe part numbers. The
96-Sample SureSelect XT HS2 DNA Reagent Kits listed in Table 54 on page 71 are compatible
with Bravo-automated processing.
• For co-analysis of RNA transcriptome variants in samples analyzed for genome variants using
a SureSelect Cancer Custom Panel Assay, Agilent recommends analyzing the samples using
the SureSelect Cancer CGP RNA probe in parallel with the SureSelect Cancer Custom Panel
probe. See page 8 for a description of the variants targeted by the SureSelect Cancer CGP RNA
Assay and see Table 59 on page 77 for CGP RNA probe ordering information.

SureSelect Cancer CGP Assay User Guide 73

Agilent SureSelect Cancer CGP Assay
User Guide

9
Reference
Reagent Kit Contents 75
SureSelect XT HS2 Index Primer Pair Information 79
SureSelect XT HS2 Index Primer Pair Sequences 80
Index Primer Pair Strip Tube and Plate Maps 84
Troubleshooting Guide 86

This section contains reference information, including Reagent Kit contents, index sequences, and
troubleshooting information for the SureSelect Cancer CGP Assay.

74
Reagent Kit Contents

SureSelect Cancer CGP Assay Kits include the component kits listed in Table 56 (for DNA + RNA
kits), Table 57 (for DNA only kits), and Table 58 (for RNA only kits). Detailed contents of each of
the multi-part component kits are shown in Table 59 through Table 65 on the following pages.
Table 56 Contents of SureSelect Cancer CGP Assay Kits for DNA + RNA analysis

Component Kit Name Storage Component Kit Part Number Usage

Condition
G9965A DNA+RNA G9966A DNA+RNA
Starter Kit Kit
(16 Samples Each) (96 Samples Each)

SureSelect XT HS2 Library Preparation Kit –20°C 5500-0146 5500-0147 DNA Library Prep
for ILM (Pre PCR)

SureSelect XT HS2 Index Primer Pairs for –20°C 5191-5687 (Index 5191-5688 (Index DNA Library Prep
ILM (Pre PCR) Pairs 1–16) Pairs 1–96)

SureSelect cDNA Module (Pre PCR) –20°C 5500-0148 5500-0149 RNA Library Prep

SureSelect XT HS2 RNA Library Preparation –20°C 5500-0150 5500-0151 RNA Library Prep
Kit for ILM (Pre PCR)

SureSelect XT HS2 Index Primer Pairs for –20°C 5191-6971 (Index 5191-5689 (Index RNA Library Prep
ILM (Pre PCR) Pairs 17–32) Pairs 97–192)

SureSelect Cancer CGP Assay Probes, DNA –80°C 5191-6990 5191-6991 DNA and RNA
& RNA Library Enrichment

SureSelect Target Enrichment Kit, ILM Hyb Room 5190-9685 (2 kits) 5190-9687 (2 kits) DNA and RNA
Module, Box 1 (Post PCR) Temperature Library Enrichment

SureSelect XT HS2 Target Enrichment Kit –20°C 5191-6686 (2 kits) 5191-6688 (2 kits) DNA and RNA
ILM Hyb Module, Box 2 (Post PCR) Library Enrichment

SureSelect Streptavidin Beads +4°C 5191-5741 (2 vials) 5191-5742 (2 vials) DNA and RNA
Library Enrichment

SureSelect DNA AMPure® XP Beads* +4°C 5191-5739 5191-5740 DNA Library

Prep/Enrichment
Purifications

SureSelect RNA AMPure® XP Beads* +4°C 5191-6670 5191-6671 RNA Library

Prep/Enrichment
Purifications

SureSelect Enzymatic Fragmentation Kit –20°C 5191-4079 Not supplied, optional DNA Library Prep
(order p/n 5191-4080
separately)

OneSeq Human Reference DNA, Female +4°C 5190-8850 Not supplied, optional Control and
(order p/n 5190-8850 unmatched
separately) reference DNA

QPCR Human Reference Total RNA –80°C 750500 Not supplied, optional Control RNA
(order p/n 750500
separately)

* AMPure, Beckman, and Beckman Coulter are trademarks or registered trademarks of Beckman Coulter, Inc. SureSelect DNA AMPure XP Beads
and SureSelect RNA AMPure XP Beads may be used interchangeably.

SureSelect Cancer CGP Assay User Guide 75

Table 57 Contents of SureSelect Cancer CGP Assay Kits for DNA analysis

Component Kit Name Storage Component Kit Part Number Usage

Condition
G9967A DNA Kit G9967B DNA Kit
(16 Samples) (96 Samples)

SureSelect XT HS2 Library Preparation Kit –20°C 5500-0146 5500-0147 DNA Library Prep
for ILM (Pre PCR)

SureSelect XT HS2 Index Primer Pairs for –20°C 5191-5687 (Index 5191-5688 (Index DNA Library Prep
ILM (Pre PCR) Pairs 1–16) Pairs 1–96)

SureSelect Cancer CGP Assay Probe, DNA –80°C 5280-0035 5280-0036 Target Enrichment

SureSelect Target Enrichment Kit, ILM Hyb Room 5190-9685 5190-9687 Target Enrichment
Module, Box 1 (Post PCR) Temperature

SureSelect XT HS2 Target Enrichment Kit –20°C 5191-6686 5191-6688 Target Enrichment
ILM Hyb Module, Box 2 (Post PCR)

SureSelect Streptavidin Beads +4°C 5191-5741 5191-5742 Target Enrichment

Capture

SureSelect DNA AMPure® XP Beads +4°C 5191-5739 5191-5740 DNA Library Prep/
Enrichment Purifications

.
Table 58 Contents of SureSelect Cancer CGP Assay Kits for RNA analysis

Component Kit Name Storage Component Kit Part Number Usage

Condition
G9968A RNA Kit G9968B RNA Kit
(16 Samples) (96 Samples)

SureSelect cDNA Module (Pre PCR) –20°C 5500-0148 5500-0149 RNA Library Prep

SureSelect XT HS2 RNA Library Preparation –20°C 5500-0150 5500-0151 RNA Library Prep
Kit for ILM (Pre PCR)

SureSelect XT HS2 Index Primer Pairs for –20°C 5191-6971(Index 5191-5689 (Index RNA Library Prep
ILM (Pre PCR) Pairs 17–32) Pairs 97–192)

SureSelect Cancer CGP Assay Probe, RNA –80°C 5191-6996 5191-6997 Target Enrichment

SureSelect Target Enrichment Kit, ILM Hyb Room 5190-9685 5190-9687 Target Enrichment
Module, Box 1 (Post PCR) Temperature

SureSelect XT HS2 Target Enrichment Kit –20°C 5191-6686 5191-6688 Target Enrichment
ILM Hyb Module, Box 2 (Post PCR)

SureSelect Streptavidin Beads +4°C 5191-5741 5191-5742 Target Enrichment

Capture

SureSelect RNA AMPure® XP Beads +4°C 5191-6670 5191-6671 RNA Library Prep/
Enrichment Purifications

SureSelect Cancer CGP Assay User Guide 76

Table 59 Contents of SureSelect Cancer CGP Assay Probe Kits

Kit Part Product Name Storage Component(s) Provided Usage

Number Condition

5191-6990 SureSelect Cancer CGP Assay Probes, –80°C 5264-1001 SureSelect Cancer CGP DNA Library Enrichment
DNA & RNA, 16 Hyb Reactions/Probe Assay Probe DNA

5191-6894 SureSelect Cancer CGP RNA Library Enrichment

Assay Probe RNA

5191-6991 SureSelect Cancer CGP Assay Probes, –80°C 5264-1002 SureSelect Cancer CGP DNA Library Enrichment
DNA & RNA, 96 Hyb Reactions/Probe Assay Probe DNA

5191-6896 SureSelect Cancer CGP RNA Library Enrichment

Assay Probe RNA

5280-0035 SureSelect Cancer CGP Assay Probe, –80°C 5264-1001 SureSelect Cancer CGP DNA Library Enrichment
DNA, 16 Hyb Reactions Assay Probe DNA

5280-0036 SureSelect Cancer CGP Assay Probe, –80°C 5264-1002 SureSelect Cancer CGP DNA Library Enrichment
DNA, 96 Hyb Reactions Assay Probe DNA

5191-6996 SureSelect Cancer CGP Assay Probe, –80°C 5191-6894 SureSelect Cancer CGP RNA Library Enrichment
RNA, 16 Hyb Reactions Assay Probe RNA

5191-6997 SureSelect Cancer CGP Assay Probe, –80°C 5191-6896 SureSelect Cancer CGP RNA Library Enrichment
RNA, 96 Hyb Reactions Assay Probe RNA

Table 60 SureSelect XT HS2 Library Preparation Kit for ILM (Pre PCR) content

Kit Component 16 Reaction Kit (p/n 5500-0146) 96 Reaction Kit (p/n 5500-0147)
End Repair-A Tailing Enzyme Mix tube with orange cap tube with orange cap
End Repair-A Tailing Buffer tube with yellow cap bottle
T4 DNA Ligase tube with blue cap tube with blue cap
Ligation Buffer tube with purple cap bottle
SureSelect XT HS2 Adaptor Oligo Mix tube with white cap tube with white cap
Herculase II Fusion DNA Polymerase tube with red cap tube with red cap
5× Herculase II Reaction Buffer with dNTPs tube with clear cap tube with clear cap

Table 61 SureSelect cDNA Module (Pre PCR) content

Kit Component 16 Reaction Kit (p/n 5500-0148) 96 Reaction Kit (p/n 5500-0149)
2X Priming Buffer tube with purple cap tube with purple cap
First Strand Master Mix* amber tube with amber cap amber tube with amber cap
Second Strand Enzyme Mix tube with blue cap bottle
Second Strand Oligo Mix tube with yellow cap tube with yellow cap

* The First Strand Master Mix contains actinomycin D. Keep the reagent in the supplied amber vial to protect the contents from exposure to light.

SureSelect Cancer CGP Assay User Guide 77

Table 62 SureSelect XT HS2 RNA Library Preparation Kit for ILM (Pre PCR) content

Kit Component 16 Reaction Kit (p/n 5500-0150) 96 Reaction Kit (p/n 5500-0151)
End Repair-A Tailing Enzyme Mix tube with orange cap tube with orange cap
End Repair-A Tailing Buffer tube with yellow cap bottle
T4 DNA Ligase tube with blue cap tube with blue cap
Ligation Buffer tube with purple cap bottle
XT HS2 RNA Adaptor Oligo Mix tube with green cap tube with green cap
Herculase II Fusion DNA Polymerase tube with red cap tube with red cap
5× Herculase II Reaction Buffer with dNTPs tube with clear cap tube with clear cap

Table 63 SureSelect XT HS2 Index Primer Pairs for ILM (Pre PCR) content

Kit Component 16 Reaction Kits* 96 Reaction Kits†

p/n 5191-5687 p/n 5191-6971 p/n 5191-5688 p/n 5191-5689
(use for DNA libraries) (use for RNA libraries) (use for DNA libraries) (use for RNA libraries)
SureSelect XT HS2 Blue 8-well strip tube Black 8-well strip tube Orange 96-well plate Blue 96-well plate
Index Primer Pairs (index pairs 1-8) AND (index pairs 17-24) AND (index pairs 1–96) (index pairs 97–192)
for ILM (Pre PCR) white 8-well strip tube red 8-well strip tube
(index pairs 9-16) (index pairs 25-32)

* See page 79 through page 80 for index pair sequence information; see page 84 for index strip position maps.
† See page 79 through page 83 for index pair sequence information; see page 85 for index plate position maps.

Table 64 SureSelect Target Enrichment Kit, ILM Hyb Module Box 1 (Post PCR) content

Kit Component 16 Reaction Kit (p/n 5190-9685) 96 Reaction Kit (p/n 5190-9687)

SureSelect Binding Buffer bottle bottle

SureSelect Wash Buffer 1 bottle bottle

SureSelect Wash Buffer 2 bottle bottle

Table 65 SureSelect XT HS2 Target Enrichment Kit, ILM Hyb Module Box 2 (Post PCR) content

Kit Component 16 Reaction Kit (p/n 5191-6686) 96 Reaction Kit (p/n 5191-6688)

SureSelect Fast Hybridization Buffer bottle bottle

SureSelect XT HS2 Blocker Mix tube with blue cap tube with blue cap

SureSelect RNase Block tube with purple cap tube with purple cap

SureSelect Post-Capture Primer Mix tube with clear cap tube with clear cap

Herculase II Fusion DNA Polymerase tube with red cap tube with red cap

5× Herculase II Reaction Buffer with dNTPs tube with clear cap tube with clear cap

SureSelect Cancer CGP Assay User Guide 78

SureSelect XT HS2 Index Primer Pair Information

The SureSelect XT HS2 Index Primer Pairs are provided pre-combined. Each member of the
primer pair contains a unique 8-bp P5 or P7 index, resulting in dual-indexed NGS libraries. One
primer pair is provided in each well of 8-well strip tubes (16 reaction kits; see Figure 7 for a map)
or of 96-well plates (96 reaction kits; see page 85 for plate maps). Each well contains a single-use
aliquot of a specific pair of forward plus reverse primers.
The nucleotide sequence of the index portion of each primer is provided in Table 67 on page 80
through Table 70 on page 83. Index sequences can also be obtained by downloading the
SureSelect XT HS2 Index Sequence Resource Excel spreadsheet from Agilent.com.
Clicking the Excel spreadsheet link from Agilent.com automatically downloads
N OTE the index spreadsheet to the default folder for downloaded files saved by your
web browser. Locate the file in the folder and open it in Microsoft Excel or another
compatible spreadsheet program. The first tab of the spreadsheet provides
instructions for use of the spreadsheet contents.

In Table 67 through Table 70 and in the downloadable Excel spreadsheet, P7 indexes are shown
in forward orientation, applicable to any of the supported Illumina platforms. P5 indexes are
shown in two orientations (forward and reverse complement) for use with different platforms and
sequencing run setup and management tools, e.g., Local Run Manager and Instrument Run
Setup. Illumina sequencing platforms and their P5 sequencing orientation are shown in Table 66.
Correct representation of the P5 index orientation in sample sheets or during sequencing run
setup is crucial to successful demultiplexing. Refer to Illumina support documentation and
resources to determine the correct P5 index orientation for your application.
Table 66 P5 index sequencing orientation by Illumina platform

P5 Index Orientation Platform

Forward NovaSeq 6000 with v1.0 chemistry
*
Reverse Complement NovaSeq 6000 with v1.5 chemistry
NextSeq 500/550/1000/2000
HiSeq 3000/4000

* Some run setup and management tools used with these platforms automatically create the reverse complement se-
quence for the P5 index sequence entered for the run. Be sure to consult Illumina’s support documentation for the
combination of platform and tools used in your pipeline to determine the correct index orientation to enter during run
setup.

SureSelect Cancer CGP Assay User Guide 79

 SureSelect XT HS2 Index Primer Pair Sequences

Table 67 SureSelect XT HS2 Index Primer Pairs 1–48, provided in orange 96-well plate or in strip tubes

Primer Index P7 Index P5 Index P5 Index Primer Index P7 Index P5 Index P5 Index
Pair # Strip Forward Forward Reverse Pair # Strip Forward Forward Reverse
Complement Complement
1 A01 CAAGGTGA ATGGTTAG CTAACCAT 25 A04 AGATGGAT TGGCACCA TGGTGCCA
2 B01 TAGACCAA CAAGGTGA TCACCTTG 26 B04 GAATTGTG AGATGGAT ATCCATCT
3 C01 AGTCGCGA TAGACCAA TTGGTCTA 27 C04 GAGCACTG GAATTGTG CACAATTC
4 D01 CGGTAGAG AGTCGCGA TCGCGACT 28 D04 GTTGCGGA GAGCACTG CAGTGCTC
5 E01 TCAGCATC AAGGAGCG CGCTCCTT 29 E04 AATGGAAC GTTGCGGA TCCGCAAC
6 F01 AGAAGCAA TCAGCATC GATGCTGA 30 F04 TCAGAGGT AATGGAAC GTTCCATT
7 G01 GCAGGTTC AGAAGCAA TTGCTTCT 31 G04 GCAACAAT TCAGAGGT ACCTCTGA
8 H01 AAGTGTCT GCAGGTTC GAACCTGC 32 H04 GTCGATCG GCAACAAT ATTGTTGC
9 A02 CTACCGAA AAGTGTCT AGACACTT 33 A05 ATGGTAGC GTCGATCG CGATCGAC
10 B02 TAGAGCTC CTACCGAA TTCGGTAG 34 B05 CGCCAATT ATGGTAGC GCTACCAT
11 C02 ATGTCAAG TAGAGCTC GAGCTCTA 35 C05 GACAATTG CGCCAATT AATTGGCG
12 D02 GCATCATA ATGTCAAG CTTGACAT 36 D05 ATATTCCG GACAATTG CAATTGTC
13 E02 GACTTGAC GCATCATA TATGATGC 37 E05 TCTACCTC ATATTCCG CGGAATAT
14 F02 CTACAATG GACTTGAC GTCAAGTC 38 F05 TCGTCGTG TCTACCTC GAGGTAGA
15 G02 TCTCAGCA CTACAATG CATTGTAG 39 G05 ATGAGAAC TCGTCGTG CACGACGA
16 H02 AGACACAC TCTCAGCA TGCTGAGA 40 H05 GTCCTATA ATGAGAAC GTTCTCAT
17 A03 CAGGTCTG AGACACAC GTGTGTCT 41 A06 AATGACCA GTCCTATA TATAGGAC
18 B03 AATACGCG CAGGTCTG CAGACCTG 42 B06 CAGACGCT AATGACCA TGGTCATT
19 C03 GCACACAT AATACGCG CGCGTATT 43 C06 TCGAACTG CAGACGCT AGCGTCTG
20 D03 CTTGCATA GCACACAT ATGTGTGC 44 D06 CGCTTCCA TCGAACTG CAGTTCGA
21 E03 ATCCTCTT CTTGCATA TATGCAAG 45 E06 TATTCCTG CGCTTCCA TGGAAGCG
22 F03 GCACCTAA ATCCTCTT AAGAGGAT 46 F06 CAAGTTAC TATTCCTG CAGGAATA
23 G03 TGCTGCTC GCACCTAA TTAGGTGC 47 G06 CAGAGCAG CAAGTTAC GTAACTTG
24 H03 TGGCACCA TGCTGCTC GAGCAGCA 48 H06 CGCGCAAT CAGAGCAG CTGCTCTG

SureSelect Cancer CGP Assay User Guide 80

Table 68 SureSelect XT HS2 Index Primer Pairs 49–96, provided in orange 96-well plate

Primer Index P7 Index P5 Index P5 Index Primer Index P7 Index P5 Index P5 Index
Pair # Strip Forward Forward Reverse Pair # Strip Forward Forward Reverse
Complement Complement
49 A07 TGAGGAGT CGCGCAAT ATTGCGCG 73 A10 AACGCATT ATAGTGAC GTCACTAT
50 B07 ATGACGAA TGAGGAGT ACTCCTCA 74 B10 CAGTTGCG AACGCATT AATGCGTT
51 C07 TACGGCGA ATGACGAA TTCGTCAT 75 C10 TGCCTCGA CAGTTGCG CGCAACTG
52 D07 AGCGAGTT TACGGCGA TCGCCGTA 76 D10 AAGGCTTA TGCCTCGA TCGAGGCA
53 E07 TGTATCAC AGCGAGTT AACTCGCT 77 E10 GCAATGAA AAGGCTTA TAAGCCTT
54 F07 GATCGCCT TGTATCAC GTGATACA 78 F10 AAGAACCT GCAATGAA TTCATTGC
55 G07 GACTCAAT GATCGCCT AGGCGATC 79 G10 CTGTGCCT AAGAACCT AGGTTCTT
56 H07 CAGCTTGC GACTCAAT ATTGAGTC 80 H10 TACGTAGC CTGTGCCT AGGCACAG
57 A08 AGCTGAAG CAGCTTGC GCAAGCTG 81 A11 AAGTGGAC TACGTAGC GCTACGTA
58 B08 ATTCCGTG AGCTGAAG CTTCAGCT 82 B11 CAACCGTG AAGTGGAC GTCCACTT
59 C08 TATGCCGC ATTCCGTG CACGGAAT 83 C11 CTGTTGTT CAACCGTG CACGGTTG
60 D08 TCAGCTCA TATGCCGC GCGGCATA 84 D11 GCACGATG CTGTTGTT AACAACAG
61 E08 AACTGCAA TCAGCTCA TGAGCTGA 85 E11 GTACGGAC GCACGATG CATCGTGC
62 F08 ATTAGGAG AACTGCAA TTGCAGTT 86 F11 CTCCAAGC GTACGGAC GTCCGTAC
63 G08 CAGCAATA ATTAGGAG CTCCTAAT 87 G11 TAGTCTGA CTCCAAGC GCTTGGAG
64 H08 GCCAAGCT CAGCAATA TATTGCTG 88 H11 TTCGCCGT TAGTCTGA TCAGACTA
65 A09 TCCGTTAA GCCAAGCT AGCTTGGC 89 A12 GAACTAAG ATACGAAG CTTCGTAT
66 B09 GTGCAACG TCCGTTAA TTAACGGA 90 B12 AAGCCATC GAGATTCA TGAATCTC
67 C09 AGTAACGC GTGCAACG CGTTGCAC 91 C12 AACTCTTG AAGCCATC GATGGCTT
68 D09 CATAGCCA AGTAACGC GCGTTACT 92 D12 GTAGTCAT AACTCTTG CAAGAGTT
69 E09 CACTAGTA CATAGCCA TGGCTATG 93 E12 CTCGCTAG GTAGTCAT ATGACTAC
70 F09 TTAGTGCG CACTAGTA TACTAGTG 94 F12 AGTCTTCA CAGTATCA TGATACTG
71 G09 TCGATACA TTAGTGCG CGCACTAA 95 G12 TCAAGCTA CTTCGTAC GTACGAAG
72 H09 ATAGTGAC TCGATACA TGTATCGA 96 H12 CTTATCCT TCAAGCTA TAGCTTGA

SureSelect Cancer CGP Assay User Guide 81

Table 69 SureSelect XT HS2 Index Primer Pairs 97–144, provided in blue 96-well plate

Primer Index P7 Index P5 Index P5 Index Primer Index P7 Index P5 Index P5 Index
Pair # Strip Forward Forward Reverse Pair # Strip Forward Forward Reverse
Complement Complement
97 A01 TCATCCTT CTTATCCT AGGATAAG 121 A04 CAGGCAGA AGACGCCT AGGCGTCT
98 B01 AACACTCT TCATCCTT AAGGATGA 122 B04 TCCGCGAT CAGGCAGA TCTGCCTG
99 C01 CACCTAGA AACACTCT AGAGTGTT 123 C04 CTCGTACG TCCGCGAT ATCGCGGA
100 D01 AGTTCATG CACCTAGA TCTAGGTG 124 D04 CACACATA CTCGTACG CGTACGAG
101 E01 GTTGGTGT AGTTCATG CATGAACT 125 E04 CGTCAAGA CACACATA TATGTGTG
102 F01 GCTACGCA GTTGGTGT ACACCAAC 126 F04 TTCGCGCA CGTCAAGA TCTTGACG
103 G01 TCAACTGC GCTACGCA TGCGTAGC 127 G04 CGACTACG TTCGCGCA TGCGCGAA
104 H01 AAGCGAAT TCAACTGC GCAGTTGA 128 H04 GAAGGTAT CGACTACG CGTAGTCG
105 A02 GTGTTACA AAGCGAAT ATTCGCTT 129 A05 TTGGCATG GAAGGTAT ATACCTTC
106 B02 CAAGCCAT GTGTTACA TGTAACAC 130 B05 CGAATTCA TTGGCATG CATGCCAA
107 C02 CTCTCGTG CAAGCCAT ATGGCTTG 131 C05 TTAGTTGC CGAATTCA TGAATTCG
108 D02 TCGACAAC CTCTCGTG CACGAGAG 132 D05 GATGCCAA TTAGTTGC GCAACTAA
109 E02 TCGATGTT TCGACAAC GTTGTCGA 133 E05 AGTTGCCG GATGCCAA TTGGCATC
110 F02 CAAGGAAG TCGATGTT AACATCGA 134 F05 GTCCACCT AGTTGCCG CGGCAACT
111 G02 ATTGATGC AGAGAATC GATTCTCT 135 G05 ATCAAGGT GTCCACCT AGGTGGAC
112 H02 TCGCAGAT TTGATGGC GCCATCAA 136 H05 GAACCAGA ATCAAGGT ACCTTGAT
113 A03 GCAGAGAC TCGCAGAT ATCTGCGA 137 A06 CATGTTCT GAACCAGA TCTGGTTC
114 B03 CTGCGAGA GCAGAGAC GTCTCTGC 138 B06 TCACTGTG CATGTTCT AGAACATG
115 C03 CAACCAAC CTGCGAGA TCTCGCAG 139 C06 ATTGAGCT TCACTGTG CACAGTGA
116 D03 ATCATGCG CAACCAAC GTTGGTTG 140 D06 GATAGAGA ATTGAGCT AGCTCAAT
117 E03 TCTGAGTC ATCATGCG CGCATGAT 141 E06 TCTAGAGC GATAGAGA TCTCTATC
118 F03 TCGCCTGT TCTGAGTC GACTCAGA 142 F06 GAATCGCA TCTAGAGC GCTCTAGA
119 G03 GCGCAATT TCGCCTGT ACAGGCGA 143 G06 CTTCACGT GAATCGCA TGCGATTC
120 H03 AGACGCCT GCGCAATT AATTGCGC 144 H06 CTCCGGTT CTTCACGT ACGTGAAG

SureSelect Cancer CGP Assay User Guide 82

Table 70 SureSelect XT HS2 Index Primer Pairs 145–192, provided in blue 96-well plate

Primer Index P7 Index P5 Index P5 Index Primer Index P7 Index P5 Index P5 Index
Pair # Strip Forward Forward Reverse Pair # Strip Forward Forward Reverse
Complement Complement
145 A07 TGTGACTA CTCCGGTT AACCGGAG 169 A10 CGCTCAGA CTAACAAG CTTGTTAG
146 B07 GCTTCCAG TGTGACTA TAGTCACA 170 B10 TAACGACA CGCTCAGA TCTGAGCG
147 C07 CATCCTGT GCTTCCAG CTGGAAGC 171 C10 CATACTTG TAACGACA TGTCGTTA
148 D07 GTAATACG CATCCTGT ACAGGATG 172 D10 AGATACGA CATACTTG CAAGTATG
149 E07 GCCAACAA GTAATACG CGTATTAC 173 E10 AATCCGAC AGATACGA TCGTATCT
150 F07 CATGACAC GCCAACAA TTGTTGGC 174 F10 TGAAGTAC AATCCGAC GTCGGATT
151 G07 TGCAATGC CATGACAC GTGTCATG 175 G10 CGAATCAT TGAAGTAC GTACTTCA
152 H07 CACATTCG TGCAATGC GCATTGCA 176 H10 TGATTGGC CGAATCAT ATGATTCG
153 A08 CAATCCGA CACATTCG CGAATGTG 177 A11 TCGAAGGA TGATTGGC GCCAATCA
154 B08 CATCGACG CAATCCGA TCGGATTG 178 B11 CAGTCATT TCGAAGGA TCCTTCGA
155 C08 GTGCGCTT CATCGACG CGTCGATG 179 C11 CGCGAACA CAGTCATT AATGACTG
156 D08 ATAGCGTT GTGCGCTT AAGCGCAC 180 D11 TACGGTTG CGCGAACA TGTTCGCG
157 E08 GAGTAAGA ATAGCGTT AACGCTAT 181 E11 AGAACCGT TACGGTTG CAACCGTA
158 F08 CTGACACA GAGTAAGA TCTTACTC 182 F11 AGGTGCTT AGAACCGT ACGGTTCT
159 G08 ATACGTGT CTGACACA TGTGTCAG 183 G11 ATCGCAAC AGGTGCTT AAGCACCT
160 H08 GACCGAGT ATACGTGT ACACGTAT 184 H11 GCCTCTCA ATCGCAAC GTTGCGAT
161 A09 GCAGTTAG GACCGAGT ACTCGGTC 185 A12 TCGCGTCA GCCTCTCA TGAGAGGC
162 B09 CGTTCGTC GCAGTTAG CTAACTGC 186 B12 GAGTGCGT TCGCGTCA TGACGCGA
163 C09 CGTTAACG CGTTCGTC GACGAACG 187 C12 CGAACACT GCATAAGT ACTTATGC
164 D09 TCGAGCAT CGTTAACG CGTTAACG 188 D12 TAAGAGTG AGAAGACG CGTCTTCT
165 E09 GCCGTAAC TCGAGCAT ATGCTCGA 189 E12 TGGATTGA TAAGAGTG CACTCTTA
166 F09 GAGCTGTA GCCGTAAC GTTACGGC 190 F12 AGGACATA TGGATTGA TCAATCCA
167 G09 AGGAAGAT GAGCTGTA TACAGCTC 191 G12 GACATCCT AGGACATA TATGTCCT
168 H09 CTAACAAG AGGAAGAT ATCTTCCT 192 H12 GAAGCCTC GACATCCT AGGATGTC

SureSelect Cancer CGP Assay User Guide 83

 Index Primer Pair Strip Tube and Plate Maps
SureSelect XT HS2 Index Primer Pairs 1-16 and 17-32 (provided with 16 reaction kits) are supplied
in sets of two 8-well strip tubes as detailed below.

Figure 7 Map of the SureSelect XT HS2 Index Primer Pairs for ILM (Pre PCR) strip tubes provid-
ed with 16 reaction kits

Index Primer Pairs 1-8 are provided in a blue strip, with pair #1 supplied in the well proximal to the
numeral 1 etched on the strip’s plastic end tab.
Index Primer Pairs 9-16 are provided in a white strip, with pair #9 supplied in the well proximal to
the numeral 9 etched on the strip’s plastic end tab.
Index Primer Pairs 17-24 are provided in a black strip, with pair #17 supplied in the well proximal
to the numeral 17 etched on the strip’s plastic end tab.
Index Primer Pairs 25-32 are provided in a red strip, with pair #25 supplied in the well proximal to
the numeral 25 etched on the strip’s plastic end tab.
When using the strip tube- supplied index primer pairs in the library preparation protocol, pierce
the foil seal of the appropriate well with a pipette tip just before pipetting the solution. If the foil
seal for any unused wells is disrupted during use, the unused wells may be re- sealed using the
provided fresh foil seal strips. The provided foil strips may also be used to re- seal used wells to
prevent index pair cross- contamination during subsequent use.
See Table 71 and Table 72 on page 85 for plate maps showing positions of the SureSelect XT
HS2 Index Primer Pairs provided with 96 reaction kits.

The SureSelect XT HS2 Index Primer Pairs are provided in single-use aliquots. To avoid
CAU TIO N cross-contamination of libraries, use each well in only one library preparation reaction. Do
not retain and re-use any residual volume for subsequent experiments.

SureSelect Cancer CGP Assay User Guide 84

Table 71 Plate map for SureSelect XT HS2 Index Primer Pairs 1-96, provided in orange plate

1 2 3 4 5 6 7 8 9 10 11 12

A 1 9 17 25 33 41 49 57 65 73 81 89

B 2 10 18 26 34 42 50 58 66 74 82 90

C 3 11 19 27 35 43 51 59 67 75 83 91

D 4 12 20 28 36 44 52 60 68 76 84 92

E 5 13 21 29 37 45 53 61 69 77 85 93

F 6 14 22 30 38 46 54 62 70 78 86 94

G 7 15 23 31 39 47 55 63 71 79 87 95

H 8 16 24 32 40 48 56 64 72 80 88 96

Table 72 Plate map for SureSelect XT HS2 Index Primer Pairs 97-192, provided in blue plate

1 2 3 4 5 6 7 8 9 10 11 12

A 97 105 113 121 129 137 145 153 161 169 177 185

B 98 106 114 122 130 138 146 154 162 170 178 186

C 99 107 115 123 131 139 147 155 163 171 179 187

D 100 108 116 124 132 140 148 156 164 172 180 188

E 101 109 117 125 133 141 149 157 165 173 181 189

F 102 110 118 126 134 142 150 158 166 174 182 190

G 103 111 119 127 135 143 151 159 167 175 183 191

H 104 112 120 128 136 144 152 160 168 176 184 192

SureSelect Cancer CGP Assay User Guide 85

Troubleshooting Guide

If recovery of gDNA from samples is low

Using excess tissue for gDNA isolation can reduce yield. Use only the amount of each specific tissue
type recommended by the gDNA isolation protocol.
Tissue sample lysis may not have been optimal during gDNA isolation. Monitor the extent of sample
lysis during the Proteinase K digestion at 56°C by gently pipetting the digestion reaction every 20–30
minutes, visually inspecting the solution for the presence of tissue clumps. If clumps are still present
after the 1-hour incubation at 56°C, add another 10 µL of Proteinase K and continue incubating at 56°C,
with periodic mixing and visual inspections, for up to two additional hours. When the sample no longer
contains clumps of tissue, move the sample to room temperature until lysis is complete for the
remaining samples. Do not over-digest. Individual samples may be kept at room temperature for up to
2 hours before resuming the protocol. Do not exceed 3 hours incubation at 56°C for any sample.

If samples contain <50 ng gDNA or total RNA

The SureSelect Cancer CGP Assay requires sample input amounts of 50 ng genomic DNA or 50 ng total
RNA for optimal performance including enabling high-confidence discovery of variant alleles down to
5% frequency. The SureSelect XT HS2 reagent system used for library preparation and target
enrichment supports use of 10–200 ng DNA or RNA input. Accordingly, libraries can be prepared from
as little as 10 ng input, with possible negative impacts on yield and sequencing coverage. If <50 DNA or
RNA is recovered for a sample in the run, review the considerations below:
If additional starting material is available, perform an additional round of gDNA or total RNA isolation for
the sample.
If library preparation is performed using 10-50 ng gDNA or total RNA, increase the pre-capture PCR cycle
number by 1 to 2 cycles (add 2 cycles for minimal 10 ng input).
Library preparation using poor-quality FFPE DNA (DIN<3) or RNA (DV200<50%) at <50 ng input is not
recommended, even when using self-optimized conditions.

If yield of pre-capture libraries is low

The library preparation protocol includes specific thawing, temperature control, pipetting, and mixing
instructions which are required for optimal performance of the highly viscous buffer and enzyme
solutions used in the protocol. Be sure to adhere to all instructions when setting up the reactions.
Ensure that the ligation master mix (see page 31) is kept at room temperature for 30–45 minutes before
use.
PCR cycle number may require optimization. Repeat library preparation for the sample, increasing the
pre-capture PCR cycle number by 1 to 2 cycles. If a high molecular weight peak (>500 bp) is observed in
the electropherogram for a sample with low yield, the DNA may be overamplified. Repeat library
preparation for the sample, decreasing the pre-capture PCR cycle number by 1 to 3 cycles.
DNA isolated from FFPE tissue samples may be over-fragmented or have modifications that adversely
affect library preparation processes. Use the Agilent NGS FFPE QC Kit to determine the precise quantity
of amplifiable DNA in the sample and allow direct normalization of input DNA amount.
Performance of the solid-phase reversible immobilization (SPRI) purification step may be poor. Verify
the expiration date for the vial of AMPure XP Beads used for purification. Adhere to all bead storage and
handling conditions recommended by the manufacturer. Ensure that the beads are kept at room
temperature for at least 30 minutes before use. Use freshly-prepared 70% ethanol for each SPRI
procedure.
DNA elution during SPRI purification steps may be incomplete. Ensure that the AMPure XP Beads are not
over-dried just prior to sample elution.

SureSelect Cancer CGP Assay User Guide 86

 If solids observed in the End Repair-A Tailing Buffer
Vortex the solution at high speed until the solids are dissolved. The observation of solids when first
thawed does not impact performance, but it is important to mix the buffer until all solutes are dissolved.

If sheared DNA pre-capture library fragment size is larger than expected in

electropherograms
Shearing may not be optimal. For intact, high-quality DNA samples, ensure that shearing is completed
using the two-round shearing protocol provided, including all spinning and vortexing steps.
Any bubbles present on the microTUBE filament may disrupt complete shearing. Spin the microTUBE for
30 seconds before the first round of shearing to ensure that any bubbles are released.

If pre-capture library fragment size is different than expected in electopherograms

FFPE DNA or FFPE RNA pre-capture libraries may have a smaller fragment size distribution due to the
presence of DNA or RNA fragments in the sample input that are smaller than the targeted
post-fragmentation size. Adhere to the DNA quality guidelines provided on page 24 and the RNA quality
guidelines provided on page 18.
DNA fragment size selection during SPRI purification depends upon using the correct ratio of sample to
AMPure XP Beads. Before removing an aliquot of beads for the purification step, mix the beads until the
suspension appears homogeneous and consistent in color and verify that you are using the bead
volume recommended for pre-capture purification on page 36.

If low molecular weight adaptor-dimer peak is present in pre-capture library

electropherograms
The presence of a low molecular weight peak, in addition to the expected peak, indicates the presence of
adaptor-dimers in the library. It is acceptable to proceed to target enrichment with library samples for
which adaptor-dimers are observed in the electropherogram at low abundance, similar to the samples
analyzed on page 38. The presence of excessive adaptor-dimers in the samples may be associated with
reduced yield of pre-capture libraries. If excessive adaptor-dimers are observed, verify that the adaptor
ligation protocol is being performed as directed on page 32. In particular, ensure that the Ligation
master mix is mixed with the sample prior to adding the Adaptor Oligo Mix to the mixture. Do not add
the Ligation master mix and the Adaptor Oligo Mix to the sample in a single step.

If yield of post-capture libraries is low

The probe used for hybridization may have been compromised. Verify the expiration date on the probe
vial or Certificate of Analysis. Adhere to the recommended storage and handling conditions. Ensure that
the probe hybridization mix is prepared immediately before use, as directed on page 41, and that
solutions containing the probe are not held at room temperature for extended periods.

If samples seep from wells during post-hybridization washes

Some users experience liquid seepage during post-hybridization wash vortexing or spinning steps,
especially when samples are processed in flexible 8-well strip tubes.
• Use of plates or strip tubes with greater rigidity, or use of a rigid tube holder support while vortexing
flexible strip tubes, may reduce the incidence of this event.
• For each protocol step that requires removal of cap strips, reseal the wells with a fresh strip of caps.
Cap deformation may result from exposure of the cap strips to the heated lid of the thermal cycler.

If post-capture library fragment size is different than expected in electropherograms

DNA fragment size selection during SPRI purification depends upon using the correct ratio of sample to
AMPure XP Beads. Before removing an aliquot of beads for the purification step, mix the beads until the
suspension appears homogeneous and consistent in color and verify that you are using the bead
volume recommended for post-capture purification on page 44.

SureSelect Cancer CGP Assay User Guide 87

 If low fraction of reads in targeted region (low percent on target) is observed
Stringency of post-hybridization washes may have been lower than required. Complete the wash steps
as directed, paying special attention to the details of SureSelect Wash Buffer 2 washes listed below:
• SureSelect Wash Buffer 2 is pre-warmed to 70°C (see page 42)
• Samples are maintained at 70°C during washes (see page 42)
• Bead suspensions are mixed thoroughly during washes by pipetting up and down and vortexing (see
page 43)
Minimize the amount of time that hybridization reactions are exposed to RT conditions during
hybridization setup. Locate a vortex and plate spinner or centrifuge in close proximity to thermal cycler
to retain the 65°C sample temperature during mixing and transfer steps (step 8 to step 9 on page 41).

If low strand specificity is observed for SureSelect Cancer CGP RNA Assay samples
Low strand-specificity can indicate issues with the RNA library preparation process including the
following:
• Contamination of the cDNA library with PCR amplicons or other non-sample derived DNA sources.
Adhere to good laboratory hygiene practices, including performance of cDNA synthesis and library
preparation steps in an area designated for Pre-PCR work.
• Contamination of the input RNA sample with gDNA. During RNA isolation, adhere to all DNA
exclusion and depletion procedures.
• Use of inappropriate cDNA synthesis or PCR amplification reagents. Only use reagents provided
with the SureSelect Cancer CGP Assay Kit to prepare RNA libraries for analysis. Do not substitute
with reagents from other kits.
• Use of expired or improperly stored cDNA synthesis reagents. In particular, ensure that the First
Strand Master Mix is used prior to the kit expiration date and is stored in the amber vial, as provided.

If a high rRNA fraction is reported for SureSelect Cancer CGP RNA Assay samples
Ribosomal RNA sequences are not included in the SureSelect Cancer CGP Probe RNA design.
Accordingly the majority of any rRNA-derived cDNAs are excluded from the library during the
hybridization and capture steps. Ensure that the hybridization and capture steps are performed at the
required stringency to minimize the presence of rRNA and other off-target sequences in the library.
Adhere to the hybridization and capture stringency precautions described in the Troubleshooting entry
for low fraction of reads in targeted region on page 88.

If expected SNV or Indel variants are not detected

SNV and Indel detection depends on performing the assay with sufficient coverage and sequencing
depth for the frequency of the variant of interest. Detection of variants present at <5% frequency may
require analysis using more than 40M reads.

If you want to perform the assay using an unsupported sample type (e.g., ctDNA or
needle aspiration sample)
Agilent has not validated the SureSelect Cancer CGP Assay using liquid biopsy or needle aspiration
samples. Use of these or any other unsupported sample types requires self-optimization of the protocol
and validation of results by the user.
If self-optimizing the assay for use with liquid biopsy ctDNA samples, Agilent recommends omitting DNA
fragmentation from the workflow.
If nucleic acids extracted from needle aspiration samples meet the assay input amount (50 ng) and
quality requirements, samples may be suitable for the assay with minor optimization of the library
preparation and target enrichment workflow segments.
Use of any unsupported sample types requires optimization of the NGS and analysis workflow
segments. Ensure that the sequencing depth is sufficient for the expected allele frequency associated
with the sample type and variant category.

SureSelect Cancer CGP Assay User Guide 88

www.agilent.com

In This Book
This guide provides instructions for the
SureSelect Cancer CGP Assay, a targeted
next-generation sequencing (NGS) solution for
interrogation of genomic and transcriptomic
features of relevance in solid tumors.

Agilent Technologies, Inc. 2023, 2024

Version D0, April 2024

*G9966-90000*
G9966-90000