Robust pipeline for variant calling in viral and bacterial system

[nekrut]

Here is an outline of a workflow that needs to be enabled in Galaxy to allow variant calling on HIV-1 datasets

• [ ] Upload all datasets for PRJNA517147
• [ ] QC with fastqc and multiqc
• [ ] Clean off Nextera adapters with trim_galore
• [ ] Map read with bwa mem against K03455.1
• [ ] Assess coverage distribution using samtools depth and visualize using Jupyter or Galaxy's built in visualization capabilities
• [ ] Downsample to ~1,000 coverage in order to run Freebayes (Freebayes hangs on datasets where coverage if too high). The downsampling is tricky and would be a good test of new workflow functionality as it will require:
  • [ ] Compute per base depth for every sample using samtools depth
  • [ ] From samtools depth compute median depth per sample. This will give one number per sample.
  • [ ] Using this number we can now calculate downsampling factor for each sample as F = 1/(median_depth_for_given_sample/1000). The downsampling is done using samtools view -s
  • [ ] Downsampling is only performed for samples where F < 1. If F >= 1 no downsampling is necessary
• [ ] Calling variants with
  • [ ] FreeBayes
  • [ ] Mutect2
  • [ ] NVC
• [ ] Normalize and resolve multiallelic sites with bcftools norm
• [ ] Convert VCF into tab-delimited data using bcftools query. Here galaxy wrapper for this tools will need to have access to VCF metadata to allow field selection

[spond]

Dear all,

The list of studies that have sequenced HIV-1 pol using Illumina is

Author (Yr)	Title	# Samples1	Genes	VL2	Region	ARV status	Subtypes3	Data Source4
Avila-Rios (2016)[18]	HIV Drug Resistance in AntiretroviralTreatment-Naïve Individuals in the LargestPublic Hospital in Nicaragua, 2011-2015	255	PR/RT	Yes	Nicaragua	Naive	B (99.6%)	SRA
Moscona (2017)[14]	Comparison between next-generation and Sanger-based sequencing for the detection of transmitted drug-resistance mutations among recently infected HIV-1 patients in Israel, 2000-2014	78	PR (76); RT (77); IN (30)	No	Israel	Naïve	B (59%); C (22%); A (14%)	SRA
Huber (2016)[19]	MinVar: A rapid and versatile tool for HIV-1 drug resistance genotyping by deep sequencing	33	PR/RT (33); IN (13)	Yes	Switzerland	NA	B (67%), A (9%), C (9%), CRF02_AG (9%)	Zenodo
Nguyen (2018)[15]	Prevalence and clinical impact of minority resistant variants in patients failing an integrase inhibitor-based regimen by ultra-deep sequencing	134	IN	No	France	Treated	B (60%), CRF02_AG (26%)	SRA
Dalmat (2018)[20]	Limited marginal utility of deep sequencing for HIV drug resistance testing in the age of integrase inhibitors	112	PR (93); RT (94); IN (38)	No	U.S.	Treated	B (95%), C (4%), D (1%)	SRA
Jair (2019)[13]	Validation of publicly-available software used in analyzing NGS data for HIV-1 drug resistance mutations and transmission networks in a Washington, DC, cohort.	42	PR (34), RT (41), IN (33)	No	U.S.	Treated	B (98%), CRF02_AG (2%)	SRA
Ode (2015)[16]	Quasispecies Analyses of the HIV-1 Near-full-length Genome With Illumina MiSeq	92	PR/RT/IN	Yes	Japan	Treated	B (61%), CRF01_AE (11%), C (11%), CRF02 (9%)	SRA
Telele (2019)[17]	Pretreatment drug resistance in a large countrywide Ethiopian HIV-1C cohort: a comparison of Sanger and high-throughput sequencing.	109	PR/RT/IN	No	Ethiopia	Naive	C (99%), D (1%)	SRA