Performs a full Nextstrain analysis on Enterovirus D68 - currently a >=700bp VP1 run and a >=6000bp full-genome run. More may be added in future.

This repository will also eventually include code to update publicly available sequences by comparing new ViPR downloads against what's already part of the most recent run, but this isn't implemented yet. However, instructions below include this already (even though it's not yet implemented).

Note that the data used for these runs is not part of this repository. You can download publicly available data using the instructions below to add to your own run, and add any of your own data, too (see notes below on this).

To run automatically-downloaded VP1 sequences with this pipeline, you'll need to install local BLAST for this analysis to work. Do this with: sudo apt-get install ncbi-blast+ (is this right?)

Download in tab delimited format all samples that are Enterovirus -> Enterovirus D -> Enterovirus D68 using ViPR's search function, with sequence length min:6400, max:8000. (Using the 'full genome' tick-box will result in fewer sequences)

BE AWARE There are two sequences with strain name US/MO/14-18949 - accession numbers KM851227 and MH708882. MH708882 is a mouse-adapted strain and should be excluded. It is in genome/config/dropped_strains.txt as US/MO/14-18949-Mouse. However, you will have to manually rename the strain in genome/genbank/genbank_sequences.fasta and genome/genbank/genbank_meta.tsv. To rename the correct sequence in the Fasta, unfortunately you will have to compare the sequence itself to the ones online, since the strain name is the same. (This is not a problem for the VP1 run, as the accession is added to the strain name, making them distinguishable. This new name for the mouse-adapted version is then in vp1/config/dropped_strains.txt.)

Download in tab delimited format all samples that are Enterovirus -> Enterovirus D -> Enterovirus D68 using ViPR's search function, without restriction on sequence length or dates. (There should be over 3,000.)

Place this file in the data folder of vp1 or genome, and include the name of that file in the Snakefile (replacing data/entero-30Jan18.tsv or similar).

Until the re-run feature is re-implemented parsed, cleaned, and BLAST-ed GenBank sequences & metadata need to go into the vp1 or genome 'genbank' folder as genbank_meta.tsv and genbank_sequences.fasta.

Place sequences and metadata from full-genome Swedish (or your own) sequences in the top-level data folder, and ensure the filenames match the swedish_seqs and swedish_meta entries in the Snakefile.

If you have other sequences & metadata (manually curated) that you'd like to add, you can include these in by replacing manual_seqs and manual_meta. These will not be blasted, so ensure they are either full-genome or contain the VP1 gene, depending on the run.

This script will allow you to look at sequences by region as well as country. The Snakefile is already set up for this kind of analysis, and region will be automatically generated for all downloaded sequences.

However, you should ensure the Swedish metadata file, and any additional 'manual' files, have an additional column called 'region' with an entry for each sample. Otherwise, no Swedish/manual sequences will have a region.

The call needs to specify the 'length' being run (vp1 or genome) and can also specify the minimum length and maximum year of sequences to be included. See comments at the beginning of the Snakefile for more explanation and examples. Notes that specifying '2018y' will include sequences UP TO 2019.0 (2018 will be the last year included).

There are also some Snakemake rules to make running some 'default' runs easier.

Note that genome runs are filtered to 200 sequences per month per country per year, while VP1 runs are filtered to 20 sequences per month per country per year. If minimum sequence length isn't specified, the defaults are >=700bp and >=6000bp for VP1 and genome runs, respectively.

Below not yet implemented! Will not work! Navigate to the enterovirus_genome folder and run snakemake "auspice/enterovirus_d68_genome_tree.json" to do a full-genome build. Initial runs may take some time, as downloading all sequences from GenBank is slow.

All accession numbers are compared, so a sequence already included in 'Swedish' or 'manual' files will not be downloaded from GenBank.

Not yet implemented! Will not work! This Snakefile is written to make adding new data from ViPR easier. Simply download the latest full collection of samples from ViPR (using the same instructions as above), place the new file in data, and replace the filename in the Snakefile. Run snakemake, and the script should automatically only download and BLAST sequences with accesssion numbers that have not previously been checked (even if they were not included in the analysis).

After adding any new sequences, the a new full Nextstrain analysis will proceed.

Currently you need already parsed, formatted, and BLASTed sequences from Genbank in the vp1 or genome 'genbank' folder as genbank_meta.tsv and genbank_sequences.fasta.

You can also have sequences to add manually, via the filenames swedish_seqs and swedish_meta in the top-level data folder or manual_seqs and manual_meta in the appropriate vp1 or genome folder. These must also be formatted and already checked that they are either full-genome or contain VP1 (depending on the run).

You can add some extra data, particularly for sequences from GenBank where more detailed information can be scraped from papers, using the file named by extra_meta. This file should be tab-delimited with five columns: accession, date, age, sex, symptom. The data will be matched up to the combined metadata (from all of the above files) by accession. Dates need to be in the format YYYY-MM-DD and sex as M or F. Age can be multiple formats, such as: 2y3m, 20m, 2y, 15d, <12y, >79y, .5-10y. Unless specified by 'm' or 'd', numbers are taken as years: 2 is 2 years and 0.3 is 0.3 years. Ranges and greater-/less-than can be in months or years: 10m-3y. Symptoms can be as you wish, but should be consistent ('afm' and 'AFM' will be two different things).

Below not yet implemented! Will not work!

In ViPR downloads as specified above, strain is not a unique identifier, as multiple segments may come from the same strain. This causes problems unique to VP1 analysis (with full-genome, this is not an issue). To handle this, in the VP1 run, the vipr_parse.py script generates new strain identifiers by combiing the original strain column with the accession number, separated by a double-underscore.

Unlike the VP1 run, strain names are not modified during the full-genome run.

ViPR sequences are not reliably labelled with the segment(s) they include (excepting whole-genome, it seems). In order to decide which sequences contain VP1, this script creates a local BLAST database against an EV-D68 reference genome VP1 sequence, then BLASTs all downloaded sequences against it.

Sequences with matches of at least 700bp are included. This was chosen because in initial runs, using >=600bp added only 47 sequences more and >=800bp lost 289 sequences. Only the matching sequence segment is taken for analysis.

When only whole-genome sequences are used, no BLASTing is done.

This Snakefile saves a copy of the most recently run parsed, downloaded ViPR file, and uses this to decide whether an accession number is 'new.' If you delete or modify the files in the 'genbank' folder that's created, then you may trigger a completely new run.