BactVar

Map reads with BWA from paired short read files to a reference genome, followed by variant calling with GATK HaplotypeCaller and gVCF best practices. A SNP-only whole-reference alignment is generated and variant effects are annotated by SnpEff.

Usage

Run in a directory of fastq (R1 and R2) files, that have already been trimmed. BactVar will assume they have the TrimGalore! suffix of _1_val_1.fq.gz/_2_val_2.fq.gz. If they don't then provide an alternative with --fastq

BactVar <reference.fasta>

Core genome extraction, phylogenetics and correction for recombination

Combine whole reference SNP alignments, extract the core genome with BactCore and generated a phylogeny with IQTREE and ClonalFrameML

cat */*aln > wga.aln 
BactCore wga.aln > core_alignment.fasta
iqtree -s core_alignment.fasta -B 1000
ClonalFrameML core_alignment.fasta.treefile core_alignment.fasta bactvarproject

Dependencies

BactVar is written in python3 and requires the following python packages:

• Biopython

BactVar has been built around bwa, GATK, snpEff etc. The most up to date versions (mid 2023) have been used:

• prokka
• Java ≥v17
• GATK (tested on v4.4.0)
• bwa (tested on 0.7.13-r1126)
• snpEff (tested on v5.1f)
• samtools (tested on v1.9)

It won't work with much earlier versions of GATK for example, so I reccomend install these versions or above.

For local installations of java I reccommend: https://pypi.org/project/install-jdk/

Citations

BWA:
Li H. and Durbin R. (2009) Fast and accurate short read alignment with Burrows-Wheeler Transform. Bioinformatics, 25:1754-60. [PMID: 19451168]

SnpEff:
"A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3.", Cingolani P, Platts A, Wang le L, Coon M, Nguyen T, Wang L, Land SJ, Lu X, Ruden DM. Fly (Austin). 2012 Apr-Jun;6(2):80-92. PMID: 22728672

BioPython:
Cock PA, Antao T, Chang JT, Chapman BA, Cox CJ, Dalke A, Friedberg I, Hamelryck T, Kauff F, Wilczynski B and de Hoon MJL (2009) Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics, 25, 1422-1423

GATK:
McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA. (2010). The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res, 20:1297-303. DOI: 10.1101/gr.107524.110.

You may also include the following for GATK:
DePristo M, Banks E, Poplin R, Garimella K, Maguire J, Hartl C, Philippakis A, del Angel G, Rivas MA, Hanna M, McKenna A, Fennell T, Kernytsky A, Sivachenko A, Cibulskis K, Gabriel S, Altshuler D, Daly M. (2011). A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet, 43:491-498. DOI: 10.1038/ng.806.

Poplin R, Ruano-Rubio V, DePristo MA, Fennell TJ, Carneiro MO, Van der Auwera GA, Kling DE, Gauthier LD, Levy-Moonshine A, Roazen D, Shakir K, Thibault J, Chandran S, Whelan C, Lek M, Gabriel S, Daly MJ, Neale B, MacArthur DG, Banks E. (2017). Scaling accurate genetic variant discovery to tens of thousands of samples bioRxiv, 201178. DOI: 10.1101/201178

Van der Auwera GA & O'Connor BD. (2020). Genomics in the Cloud: Using Docker, GATK, and WDL in Terra (1st Edition). O'Reilly Media.

PROKKA:
Seemann T. Prokka: rapid prokaryotic genome annotation Bioinformatics 2014 Jul 15;30(14):2068-9. PMID:24642063