# Genetic-Mechanisms-in-Duodenal-Polyposis A collection of scripts for running variant callers to identify somatic mutations as detailed in forthcoming research paper: Thomas et al., Genetic Mechanisms in Duodenal Polyposis.

Analysis was performed by running five separate variant callers then comparing their outputs. The five variant callers were:

• GATK UnifiedGenotyper (version 3.3.0)
• SAMTools (version 0.1.19)
• somaticSniper (version 1.0.4)
• varScan2 (version 2.4.0)
• simpleSnpCaller (in-house script)

We provide a shell script for each caller to illustrate the parameter settings used in each case:

runUnifiedGenotyper.sh

UnifiedGenotype is one of the variant callers provided by the Genome Analysis Toolkit, [GATK] (https://software.broadinstitute.org/gatk/).

The provided shell script runUnifiedGenotyper.sh runs the GATK tools UnifiedGenotyper followed by VariantFiltration. GATK is written in Java and will require Java runtime to be installed co-installed.

Usage, e.g., ./runUnifiedGenotyper.sh \ normal.bam \ tumour.bam \ ucsc.hg19.fasta \ All_Exon_50mb.bed \ dbsnp_138.hg19.vcf \ /share/apps/GATK_3.3.0

Where input parameters are:

• BAM of normal sample.
• BAM of tumour sample.
• Path to human reference hg19 in fasta format.
• Path to bed file describing target regions.
• Path to vcc file representing dbSNP (version 138 used).
• Path of GATK distribution.

References:

• The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA, 2010 GENOME RESEARCH 20:1297-303 Article
• A framework for variation discovery and genotyping using next-generation DNA sequencing data 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 NATURE GENETICS 43:491-498 Article
• From FastQ Data to High-Confidence Variant Calls: The Genome Analysis Toolkit Best Practices Pipeline Van der Auwera GA, Carneiro M, Hartl C, Poplin R, del Angel G, Levy-Moonshine A, Jordan T, Shakir K, Roazen D, Thibault J, Banks E, Garimella K, Altshuler D, Gabriel S, DePristo M, 2013 CURRENT PROTOCOLS IN BIOINFORMATICS 43:11.10.1-11.10.33 Article

runSamtools.sh

Samtools is a toolkit of programs for handling and manipulating SAM and BAM format files including variant calling.

The provided shell script runSamtools.sh runs samtools mpileup on the supplied data and then bcftools view on the resulting output. A final step uses igvtools index, part of the IGVTools toolkit, to index the resulting vcf outfile.

e.g., ./runSamtools.sh normal.bam tumour.bam ucsc.hg19.fasta Where input parameters are:

• BAM of normal sample.
• BAM of tumour sample.
• Path to human reference hg19 in fasta format.

References

• Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., Marth G., Abecasis G., Durbin R. and 1000 Genome Project Data Processing Subgroup (2009) The Sequence alignment/map (SAM) format and SAMtools. Bioinformatics, 25, 2078-9. PMID: 19505943
• Li H. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics. 2011 Nov 1;27(21):2987-93. Epub 2011 Sep 8. PMID: 21903627
• Li H. Improving SNP discovery by base alignment quality. Bioinformatics. 2011 Apr 15;27(8):1157-8. doi: 10.1093/bioinformatics/btr076. Epub 2011 Feb 13. PMID: 21320865

runSomaticSniper.sh

SomaticSniper is a dedicated somatic caller of single nucleotide variants.

The provided script runSomaticSniper.sh runs bam-somaticsniper followed by igvtools index, part of the IGVTools toolkit, to index the resulting vcf outfile.

e.g., ./runSomaticSniper.sh normal.bam tumour.bam ucsc.hg19.fasta Where input parameters are:

• BAM of normal sample.
• BAM of tumour sample.
• Path to human reference hg19 in fasta format.

Reference

• Larson, D.E., et al., (2011) SomaticSniper: identification of somatic point mutations in whole genome sequencing data. Bioinformatics, 28(3):311-317 Article

runVarScan.sh

VarScan2 is a toolkit of programs that includes the somatic caller somatic.

The provided shell script runVarScan.sh first uses SAMTools to create pileup outputs of each BAM, followed by the VarScan somatic command to create an indexed vcc outfile.

e.g., ./runVarScan.sh \ normal.bam \ tumour.bam \ ucsc.hg19.fasta \ /share/apps/

Where input parameters are:

• BAM of normal sample.
• BAM of tumour sample.
• Path to human reference hg19 in fasta format.
• Path to VarScan.jar file.

Reference

• VarScan 1: Koboldt DC, Chen K, Wylie T, Larson DE, McLellan MD, Mardis ER, Weinstock GM, Wilson RK, & Ding L (2009). VarScan: variant detection in massively parallel sequencing of individual and pooled samples. Bioinformatics (Oxford, England), 25 (17), 2283-5 PMID: 19542151 VarScan 2: Koboldt, D., Zhang, Q., Larson, D., Shen, D., McLellan, M., Lin, L., Miller, C., Mardis, E., Ding, L., & Wilson, R. (2012). VarScan 2: Somatic mutation and copy number alteration discovery in cancer by exome sequencing Genome Research DOI: 10.1101/gr.129684.111

runSimpleSnpCaller.sh

The provided simpleSnpCaller.pl perl script is a simple in-house program for identifying single nucleotide variants with respect to reference. Output is complete list of all variants without filtering (beyond specifying minimum coverage depth) to give a baseline for comparing true variant callers.

The supplied wrapper shell script runSimpleSnpCaller.sh uses SAMTools to create pileup outputs of each BAM. It then runs simpleSnpCaller.pl on each output, followed by igvtools index. Lastly the resulting vcf files are merged into a single file using bgzip, tabix part of the htslib distribution, followed by vcf-merge, part of VCFTools. Finally the resultant vcc file is indexed with igvtools index.

e.g., ./runSimpleSnpCaller.sh normal.bam tumour.bam ucsc.hg19.fasta Where input parameters are:

• BAM of normal sample.
• BAM of tumour sample.
• Path to human reference hg19 in fasta format.

References for third party tools used

IGVTools

• James T. Robinson, Helga Thorvaldsdóttir, Wendy Winckler, Mitchell Guttman, Eric S. Lander, Gad Getz, Jill P. Mesirov. Integrative Genomics Viewer. Nature Biotechnology 29, 24–26 (2011) [Article] (http://www.nature.com/nbt/journal/v29/n1/abs/nbt.1754.html)
• Helga Thorvaldsdóttir, James T. Robinson, Jill P. Mesirov. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Briefings in Bioinformatics 14, 178-192 (2013). Article

Samtools

• Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., Marth G., Abecasis G., Durbin R. and 1000 Genome Project Data Processing Subgroup (2009) The Sequence alignment/map (SAM) format and SAMtools. Bioinformatics, 25, 2078-9. PMID: 19505943
• Li H. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics. 2011 Nov 1;27(21):2987-93. Epub 2011 Sep 8. PMID: 21903627
• Li H. Improving SNP discovery by base alignment quality. Bioinformatics. 2011 Apr 15;27(8):1157-8. doi: 10.1093/bioinformatics/btr076. Epub 2011 Feb 13. PMID: 21320865