Journal of Microbiological Methods 127 (2016) 141–145

Contents lists available at ScienceDirect

Journal of Microbiological Methods

journal homepage: www.elsevier.com/locate/jmicmeth

Comparison of microbial DNA enrichment tools for metagenomic whole

genome sequencing☆
Matthew Thoendel a, Patricio R. Jeraldo b,c, Kerryl E. Greenwood-Quaintance d, Janet Z. Yao b, Nicholas Chia b,c,
Arlen D. Hanssen e, Matthew P. Abdel e, Robin Patel a,d,⁎
a
Division of Infectious Diseases, Department of Medicine, Mayo Clinic, Rochester, MN, USA
b
Center for Individualized Medicine, Mayo Clinic, Rochester, MN, USA
c
Department of Surgery, Mayo Clinic, Rochester, MN, USA
d
Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA
e
Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA

a r t i c l e i n f o a b s t r a c t

Article history: Metagenomic whole genome sequencing for detection of pathogens in clinical samples is an exciting new area for
Received 28 March 2016 discovery and clinical testing. A major barrier to this approach is the overwhelming ratio of human to pathogen
Received in revised form 23 May 2016 DNA in samples with low pathogen abundance, which is typical of most clinical specimens. Microbial DNA en-
Accepted 24 May 2016
richment methods offer the potential to relieve this limitation by improving this ratio. Two commercially avail-
Available online 26 May 2016
able enrichment kits, the NEBNext Microbiome DNA Enrichment Kit and the Molzym MolYsis Basic kit, were
Keywords:
tested for their ability to enrich for microbial DNA from resected arthroplasty component sonicate fluids from
Metagenomics prosthetic joint infections or uninfected sonicate fluids spiked with Staphylococcus aureus. Using spiked uninfect-
Whole genome sequencing ed sonicate fluid there was a 6-fold enrichment of bacterial DNA with the NEBNext kit and 76-fold enrichment
Enrichment with the MolYsis kit. Metagenomic whole genome sequencing of sonicate fluid revealed 13- to 85-fold enrich-
Clinical samples ment of bacterial DNA using the NEBNext enrichment kit. The MolYsis approach achieved 481- to 9580-fold en-
Pathogen detection richment, resulting in 7 to 59% of sequencing reads being from the pathogens known to be present in the samples.
These results demonstrate the usefulness of these tools when testing clinical samples with low microbial burden
using next generation sequencing.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction this technology in the diagnosis of neuroleptospirosis in a 14 year old

boy (Wilson et al., 2014), as well the identification of multiple viruses
Next-generation sequencing is a powerful tool used for a wide range in cases of encephalitis (Quan et al., 2010; Hoffmann et al., 2015;
of applications, including environmental and human microbiome anal- Naccache et al., 2015) and detection of pathogens associated with diar-
ysis, profiling of cancer cells, gene expression analysis, and sequencing rhea (Zhou et al., 2015; Moore et al., 2015).
of human genomes for variants related to diseases. One exciting and Perhaps the largest barrier to the promise of WGS-based infectious
expanding area of investigation is the use of metagenomic whole ge- diseases diagnosis is the inability to meaningfully enrich the yield of
nome sequencing (WGS) for diagnosing infection. The potential to de- non-human DNA from human samples. Without the ability to target
tect and identify causative organisms that are difficult to find by DNA sequencing, low microbial burden means that the overwhelming
conventional methods in an unbiased way, as well as provide insight majority of DNA sequenced then comes from host cells rather than
into the characteristics important for clinical management, such as anti- the pathogen(s). For example, only 0.0046% of reads (475 out of
biotic resistance and virulence, has garnered great interest in clinical 10,196,620) came from Leptospira species in the above-mentioned
medicine (Goldberg et al., 2015). This was highlighted by the use of neuroleptospirosis case (Wilson et al., 2014), and only 0.0012% of
reads (1612 out of 134,068,968 reads) were attributable to Astrovirus
☆ Research reported in this publication was supported by the National Institutes of in an encephalitis case thought to be caused by this virus (Naccache
Health under award number R01 AR056647. N.C. is also supported by RO1 CA179243 et al., 2015). While bioinformatic tools exist to help identify and remove
and R.P. is also supported by R01 AI91594. The content is solely the responsibility of the human reads (Zhang et al., 2015; Ames et al., 2013), greater sequencing
authors and does not necessarily represent the official views of the National Institutes of depths are necessary to obtain enough pathogen reads to identify path-
Health.
⁎ Corresponding author at: Division of Clinical Microbiology, Mayo Clinic, 200 1st St SW,
ogens and extract useful information (e.g., to assess resistance, viru-
Rochester, MN 55905, USA. lence, strain-type). This increased sequencing depth can quickly
E-mail address: patel.robin@mayo.edu (R. Patel). escalate the costs of sequencing.

http://dx.doi.org/10.1016/j.mimet.2016.05.022
0167-7012/© 2016 Elsevier B.V. All rights reserved.
142 M. Thoendel et al. / Journal of Microbiological Methods 127 (2016) 141–145

Commercial tools have emerged which are designed to address this experiments. Three samples meeting the Infectious Diseases Society
problem by enriching for microbial DNA. One method, New England of America definition of PJI (Osmon et al., 2013), were selected for WGS
Biolab's NEBNext Microbiome DNA Enrichment kit, takes advantage of analysis based on the additional criteria of being monomicrobial, having
human and other higher order eukaryotic DNA having high CpG meth- relatively high bacterial load (N100 CFUs/10 ml sonicate fluid), and
ylation rates. By using the methylated CpG-specific binding protein lacking a sinus tract.
MBD2 fused to a human IgG Fc fragment, human DNA is selectively
bound and separated using Protein A-bound magnetic beads (Feehery 2.2. DNA purification and enrichment
et al., 2013). An alternative approach, utilized by Molzym's MolYsis
kit, is to selectively lyse human cells using chaotropic reagents and de- DNA isolation was performed using the Mobio BiOstic Bacteremia
grade any released DNA with DNase prior to extraction of DNA from mi- DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA). Microbial DNA
croorganisms. Both techniques have been shown to be effective at enrichment was performed either before DNA isolation using the
enriching microbial DNA (Feehery et al., 2013; Zheng et al., 2014; MolYsis Basic5 kit (Molzym, Bremen, Germany) or after DNA isolation
Benitez-Paez et al., 2013; Gebert et al., 2008; Handschur et al., 2009; using the NEBNext Microbiome DNA Enrichment Kit (New England
Hansen et al., 2009; Horz et al., 2008; Loonen et al., 2012; Votintseva BioLabs, Ipswich, MA), per the manufacturers' protocols. Agencourt
et al., 2015), however no direct comparisons between the approaches Ampure XP beads (Beckman Coulter, Brea, CA) were used to clean up
have been reported. Additionally, previous studies using the MolYsis DNA following the NEBNext Microbiome DNA Enrichment Kit per the
kit focused on improving techniques such as real-time PCR, whereas manufacturer's recommendations to remove binding buffer reagents
use of these methods for WGS, where microbial DNA enrichment that could interfere with subsequent steps.
could be even more useful, has been less studied (Votintseva et al.,
2015). Other methods such as host cell lysis with detergents (Hasan 2.3. Spiked uninfected sonicate fluid experiments
et al., 2016) or ox bile (Zhou & Pollard, 2012) and immunoprecipitation
of DNA with inactive methyl-specific restriction endonucleases (Barnes Sonicate fluid from four clinical samples classified as uninfected
et al., 2014; Liu et al., 2016) have also been reported, but are not avail- were pooled and divided into 500 μl aliquots. S. aureus was suspended
able as commercial products. in Ringer's solution and a total of 107 CFUs were spiked into the pool.
Whole genome amplification (WGA) by multiple displacement am- This inoculum, while larger than expected in clinical samples, was se-
plification (MDA) is one technique used to amplify DNA for methods lected to allow for reliable quantification. Unspiked sonicate fluid and
such as library preparation for next generation sequencing. MDA S. aureus in Ringer's solution alone were included as controls. DNA
typically uses the high-fidelity phi29 polymerase combined with ran- was extracted with or without pretreatment with the MolYsis kit. Ali-
dom hexamer primers to amplify DNA in a non-PCR based isothermal quots of DNA (400 ng) from spiked sonicate fluid were collected from
reaction (Dean et al., 2001). WGA is particularly useful in situations untreated samples for subsequent enrichment using the NEBNext DNA
where very little DNA is present, such as single cell sequencing or low microbiome kit. Total DNA concentration was measured using a Qubit
biomass environmental samples (Hannemann et al., 2011; Rodrigue 2.0 Fluorometer (Thermo Fisher Scientific Inc., Waltham, MA). The
et al., 2009; Yilmaz et al., 2010). WGA does, however, have drawbacks, percent of bacterial DNA was calculated by measuring the concentration
as amplification bias and contaminant DNA in reagents has been ob- of S. aureus DNA by real-time PCR and dividing by the total measured
served (Yilmaz et al., 2010; Blainey & Quake, 2011; de Bourcy et al., DNA. Experiments were performed in triplicate. Statistical significance
2014; Probst et al., 2015). amongst groups was calculated using a Kruskal Wallis test and
Prosthetic joint infection (PJI) is a devastating complication of total Wilcoxon rank sum test to directly compare groups.
joint arthroplasty (Tande & Patel, 2014). Targeted treatment requires
identification of the causative pathogen(s), which can be challenging 2.4. Real-time PCR quantification of S. aureus DNA
(Osmon et al., 2013). Low organism burden, previous antibiotic treat-
ment, polymicrobial infection, and infection by fastidious organisms Real-time PCR was performed using a Roche LightCycler (Roche
all complicate the detection and identification of pathogens. WGS as a Diagnostics, Indianapolis IN) with the Roche LightCycler SYBR Green
diagnostic tool has the potential to mitigate many of these factors; how- FastStart kit (Roche Applied Science, Penzberg, Germany). Forward
ever, the low microbial burden in these infections remains a challenge. primer 5′-TGGAGAGTTTGATCCTGGCTCAG-3′ and reverse primer 5′-
This situation is not unlike that in other serious infections, such as cen- TACCGCGGCTGCTGGCAC-3′ targeting the 16S ribosomal RNA gene
tral nervous system infection, and endocarditis and other endovascular were used to amplify microbial DNA. Following DNA purification, 2 μl
infections. of template DNA was added to 20 μl total volume reactions. LightCycler
Herein, microbial DNA enrichment tools were tested for their conditions consisted of a five minute 95 °C preincubation, 40 cycles of
ability to improve bacterial DNA yields for sequencing, using clinical amplification at 95 °C for 10 s, 62 °C for 15 s, and 72 °C for 30 s, and a
samples from patients with PJI. Results should inform the ideal use of melting curve analysis of 65 °C to 95 °C at 0.10 °C/s. Serial dilutions of
metagenomics approaches to diagnose bacterial infection. S. aureus genomic DNA (IDRL-6169) were used to create standard
curves for quantification of S. aureus DNA. Negative controls, consisting
2. Materials and methods of water, were also used.

2.1. Samples 2.5. Metagenomic whole genome sequencing

Samples were collected under the Mayo Clinic Institutional All DNA for WGS was amplified using the Illustra GenomiPhi V2
Review Board protocol 10–005,574. Sonicate fluid samples were whole genome amplification kit (GE Healthcare Bio-Sciences, Pitts-
prepared from resected prosthetic hip and knee components in burgh, PA) to obtain sufficient amounts of DNA for library preparation.
Mayo Clinic's Clinical Microbiology Laboratory using previously- MolYsis pretreatment routinely resulted in insufficient amounts of
described vortexing/sonication methods (Trampuz et al., 2007; DNA for library preparation without WGA, even for kits such as Nextera
Piper et al., 2009). Negative sonicate fluid samples were selected XT which require as little as 1 ng of DNA. Paired-end libraries were
which had no clinical, laboratory, pathological, or microbiological prepared using the NEBNext Ultra DNA Library Prep Kit (New England
findings suggesting infection. A methicillin-resistant Staphylococcus BioLabs) by the Mayo Clinic Medical Genome Facility. Samples were
aureus strain (IDRL-6169) previously isolated from a patient with sequenced with the Illumina HiSeq 2500 in rapid run mode with
PJI (Vergidis et al., 2011), was used for the spiked sonicate fluid 2 × 250 bp reads. Samples were either run as single samples or
 M. Thoendel et al. / Journal of Microbiological Methods 127 (2016) 141–145 143

multiplexed with 2 or 6 samples per lane, based on the number of sam- sonicate samples was 0.27 ng/μl in the unenriched samples and
ples available to sequence at a time. 0.25 ng/μl in the MolYsis treated samples. Unspiked sonicate fluid con-
trols contained a calculated 0.03% bacterial DNA (6.8 pg/μl), indicating
2.6. WGS data analysis minimal influence of any bacterial DNA that may be present. The per-
cent of bacterial DNA was significantly different between the unen-
Paired-end reads were pre-processed using seqtk (version 1.0-r82, riched and both enriched samples (p = 0.0495).
[https://github.com/lh3/seqtk]), Trimmomatic (version 0.35) (Bolger The enrichment methods were also tested with sonicate fluids
et al., 2014) and the Livermore Metagenomics Analysis Toolkit from clinical PJI cases. These infections typically have bacterial loads
(LMAT) (Ames et al., 2013) with its pre-processing scripts. BioBloom much lower than those in the spiked sonicate fluids studied. Three
tools was used to pre-filter human and PhiX reads (Chu et al., 2014). monomicrobial PJI sonicate samples were selected, based on their hav-
Taxonomy for individual reads was assigned using LMAT with the ing a high microbial burden of a single pathogen. These samples had
kML + Human.v4-14.20.g10.db database, which attempts to make a previously been evaluated in the Mayo Clinic Clinical Microbiology
taxonomic assignment to all reads present. Reads assigned to the Laboratory and, by culture, only yielded S. aureus, Staphylococcus
known pathogen's genus group (including at the species, strains, and epidermidis, and Enterococcus faecalis from the respective samples. The
species' mobile genetic elements) were considered as being from the samples were purified as in the spiked experiments with either no en-
known pathogen. The percent of reads from the pathogen was calculat- richment, MolYsis pretreatment, or enrichment with the NEBNext
ed by dividing the number of assigned pathogen reads by the total num- Microbiome kit. Following WGA and metagenomic sequencing, LMAT
ber of reads prior to pre-processing. (Ames et al., 2013) was used to assign taxonomy to each read in order
to determine the relative amount of sequences that came from the
3. Results known PJI pathogen for each infection. All reads that were assigned to
the known pathogen's genus were included in the pathogen read calcu-
Pooled sonicate fluid from prostheses resected due to aseptic failure lations as this tool is unable to reliably classify the majority of reads to
was spiked with S. aureus and used as a tool to compare the ability of the the species level for some groups (e.g., Enterococcus species and
different methods studied to enrich for bacterial DNA. Spiked sonicate coagulase-negative Staphylococcus species) and therefore assigns them
fluid underwent DNA extraction alone, with pretreatment with MolYsis to the lowest common ancestor, which is at the genus level. WGS
prior to DNA extraction, or with the NEBNext Microbiome DNA enrich- analysis revealed a pattern of enrichment similar to that of the spiked
ment kit after DNA extraction. The percent of S. aureus DNA was then sonicate fluids. Without enrichment, 0.0062 to 0.016% of reads were
determined by measuring the amount of S. aureus DNA by real-time from the known pathogens (Table 1). Enrichment with the NEBNext mi-
PCR in relation to the total DNA concentration. crobial DNA enrichment kit improved this yield to 0.18 to 0.53%,
DNA extraction of spiked sonicate fluid without enrichment yielded representing a 13- to 85-fold enrichment of bacterial DNA. As with the
1.1% of the total DNA being from S. aureus (Fig. 1). Removal of human spiked samples, MolYsis treatment resulted in higher enrichment,
DNA with the NEBNext microbial DNA enrichment kit improved the rel- with 7.0 to 59.4% of reads being from the known pathogen, representing
ative amount of S. aureus DNA to 6.1%, representing a 5.7-fold enrich- a 481- to 9580-fold increase over the unenriched sample. Reads were
ment of bacterial DNA. Treatment of samples with the MolYsis kit mapped to the species' representative genome to confirm uniform cov-
prior to DNA extraction resulted in a significantly higher proportion of erage of reads (Fig. S1). Notably, other bacterial and viral species were
S. aureus DNA concentration at 81%, representing a 76-fold increase in also detected in the samples (Table S1), particularly Pseudomonas, Strep-
bacterial DNA. The average bacterial DNA concentration in the spiked tococcus, and Propionibacterium species, a finding consistent with previ-
ous reports of common contaminants in metagenomic studies of low
biomass samples (Salter et al., 2014; Laurence et al., 2014). Similar
types of contamination were also found in negative controls consisting
of the WGA kit alone with no template added (Table S1), albeit at higher
levels due to the lack of template DNA to compete with contaminant

Table 1
Effect of enrichment methods by metagenomic whole genome sequencing. PJI samples
underwent enrichment with either MolYsis or NEBNext microbiome kits prior to
metagenomic WGS sequencing. “% of reads indicates” the percentage of all assigned reads
attributable to the known pathogen genus as assigned by LMAT relative to the total num-
ber of reads. Read numbers are reported in parentheses. “Enrichment factor” indicates the
fold increase of percent of assigned reads compared to the unenriched sample.

No enrichment NEBNext MolYsis

microbiome enrichment
DNA enrichment

S. aureus PJI
% of reads 0.016% 0.21% 7.7%
(4158 of 25,609,460) (350,625 of (2,286,890 of
169,981,133) 29,530,730)
Enrichment factor 13× 481×
S. epidermidis PJI
% of reads 0.0071% 0.18% 7.0%
(1682 of 23,606,476) (133,680 of (2,268,087 of
74,544,475) 32,184,381)
Fig. 1. Enrichment of S. aureus DNA in spiked sonicate fluid samples. Culture-negative
Enrichment factor 25× 986×
sonicate fluid was spiked with S. aureus prior to enrichment with MolYsis or NEBNext
E. faecalis PJI
microbiome DNA enrichment kits and DNA purification. Unspiked sonicate fluid and
% of reads 0.0062% 0.53% 59.4%
S. aureus in Ringer's solution were included as controls. Percent bacterial DNA was
(1671 of 26,949,030) (497,206 of (16,407,878 of
calculated by comparing the S. aureus DNA content by real-time PCR results for S. aureus
94,522,959) 27,643,294)
16S ribosomal RNA gene to total DNA in the sample as measured using a Qubit
Enrichment factor 85× 9580×
fluorometer.
144 M. Thoendel et al. / Journal of Microbiological Methods 127 (2016) 141–145

DNA. This suggests that the primary source for these contaminant reads in order to obtain sufficient quantities of DNA for library preparation,
is the WGA kit itself. a step that could alter proportions of DNA within samples (Probst
et al., 2015). WGA has been shown to introduce bias based on GC con-
4. Discussion tent (Yilmaz et al., 2010; Probst et al., 2015; Direito et al., 2014), DNA
fragment size (Direito, 2014), and relative starting abundance
The very low relative abundance of bacterial to human DNA in many (Raghunathan et al., 2005). While these studies have evaluated the
clinical specimens deriving from patients with infection presents a biases introduced in analysis of microbial communities, the impact of
unique challenge when using WGS to detect and identify pathogens. WGA on human to bacterial DNA ratios remains unclear. However, the
We sought to compare the effectiveness of two commercially available bias against low abundance DNA raises the possibility of exaggerated ra-
kits in enriching for bacterial DNA. Whether testing uninfected sonicate tios of microbial DNA before and after enrichment. Despite this, the sim-
fluid spiked with S. aureus or PJI samples harboring known pathogens, ilar trends observed using spiked sonicate fluid samples, which did not
we observed that both techniques achieved the goal of enriching for undergo WGA, are reassuring that the WGS results are not simply due to
bacterial DNA, however the MolYsis method was more effective in this bias.
achieving this goal, with an enrichment of nearly 500- to 10,000-fold Evaluation of the metagenomic WGS results reveals reads from a va-
achieved in clinical PJI samples based on metagenomic WGS. riety of species beyond the known pathogens identified by culture
Microbial DNA enrichment methods such as these are powerful tools (Table S1). This can be largely tracked to the WGA kit as no template
for the identification and characterization of pathogenic or commensal controls contained the same species considered to be contaminants in
bacteria. By increasing the relative amount of bacterial DNA, one can PJI samples. Further studies evaluating and comparing WGA kits are
go from barely detecting an organism of interest to obtaining enough needed. This serves as an example of the caution that must be exercised
sequencing reads to assemble nearly complete genomes at sufficient when interpreting metagenomic sequencing data and the importance of
depths to carry out additional characterization such as SNP analysis, an- controls for these methods, particularly with the increasing interest of
timicrobial resistance or virulence prediction or strain-typing. This has these approaches in clinical diagnostics.
the potential to take WGS approaches beyond pathogen detection to Investigators looking at ways to enrich for microbial DNA must take
providing information useful in treatment decisions, such as selection many factors into consideration when choosing the method most ap-
of the most appropriate antibiotic (Bradley et al., 2015; Stoesser et al., propriate for their studies. The type of sample (e.g., solid tissue versus
2013), and to assessing modes of transmission. liquid), most likely pathogen(s) of interest, cost, and required extent
The studied enrichment methods are not without their limitations. of enrichment must all be carefully considered. It should be stressed
There are limited studies regarding the extent to which bacterial DNA that the enrichment factor is highly dependent on the starting relative
is removed during the protocols. Horz et al. tested caries and periodon- content of bacterial DNA. If the same amount of host DNA is removed,
tal samples after MolYsis extraction and reported a wide range of 3.3 to a sample with lower microbial content will have a higher fold-
100% of bacterial DNA remaining after extraction, as measured by16S enrichment than the same sample with higher initial microbial DNA,
rRNA gene quantitation (Horz et al., 2010). With the MolYsis approach i.e. if microbial DNA made up 1% of the total DNA prior to enrichment,
there are questions as to whether bacteria with weak or absent cell then 500-fold enrichment is not possible.
walls (e.g., Mycoplasma, Ureaplasma, or Chlamydia species), or those In conclusion, both methods tested were effective at enriching for
previously exposed to cell wall-targeting antibiotics, would be lysed microbial DNA, although the MolYsis kit provided for the greater enrich-
and removed by the technique (Horz et al., 2010). While many bacterial ment of the two. We also show that PJI can be diagnosed using a
and fungal species have been reported as being detectable after MolYsis metagenomic strategy.
purification (Gebert et al., 2008; Horz et al., 2008; Votintseva et al., Supplementary data to this article can be found online at http://dx.
2015; Meurs et al., 2011; Xu et al., 2012), these studies have been qual- doi.org/10.1016/j.mimet.2016.05.022.
itative in nature without any measurement of how much microbial DNA
might be lost. Studies examining effects on mixed communities would Acknowledgements
certainly be beneficial, since biases are likely introduced with this tech-
nique. MBD2-Fc proteins utilized by the NEBNext microbiome kit also We thank the Mayo Clinic Center for Individualized Medicine,
lack reported data on the impact on microbial DNA recovery. While Mr. Bruce Eckloff and the Mayo Clinic Medical Genome Facility for assis-
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fungi also methylate cytosines at variable rates, depending on the spe-
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