SNP calling pipeline

Status: Alpha

Authors: Freerk van Dijk, Morris Swertz

Based on ​Broad GATK pipeline.

To perform the analysis as fast and good as possible the pipeline has been divided into several small processes. These processes are all numbered and can be found below, including commands, input and output files starting with pre-alignment and ending with variation calling & filtering.

Simplified Overview

This simplified overview this schema hides intermediate sort and indexing steps and only shows data inputs/outputs first time they occur.

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Workflow 1: genome reference file creation

This workflow creates reference files per chromosome including:

• genome, dbsnp and indel vcfs per chromosome
• realign targets for faster realignment target creation
• index files for samtools and bwa

Workflow inputs:

• genome.chr.fa - downloaded from genome supplier (now hg19)
• dbsnpXYZ.rod - downloaded reference SNPs from dbsnp (now 129)
• indelsXYZ.vcf - downloaded reference indels from 1KG

Workflow outputs:

• genome.chr.fa - cleaned headers
• genome.chr.fa.fa - index for samtools
• genome.chr.fa.<format> - multilple index files for bwa
• dbsnpXYZ.chr.rod - split per chromosome
• indelsXYZ.chr.vcf - split per chromosome
• genome.chr.realign.intervals - targets for realignment

clean-fasta-headers

Clean headers to only have '1' instead of Chr1, etc

tool:
inputs:	genome.chr.fa
outputs:	genome.chr.fa
doc:	internally developed

split-vcf-chr for dbsnp and indels

Split vcf per chromosome

tool:
inputs:	dbsnpXYZ.rod, indelsXYZ.vcf
outputs:	dbsnpXYz.chr.rod, indelsXYZ.vcf
doc:

Discussion:

Can we use ​http://vcftools.sourceforge.net/options.html ?

vcftools --vcf indelsXYZ.vcf --chr <i> --recode --out indelsXYZ.chr

index-chromosomes

Index reference sequence for each chromosome in the FASTA format

tool:	samtools faidx
input:	genome.chr.fa
output:	genome.chr.fa.fai
doc:	​http://samtools.sourceforge.net/samtools.shtml#3

bwa-index-chromosomes

Index reference sequence for each chromosome for bwa alignment

tool:	bwa index -a IS
input:	genome.chr.fa
output:	genome.chr.fa.xyz
doc:	​http://bio-bwa.sourceforge.net/bwa.shtml#3

RealignerTargetCreator

Generate realignment targets for known sites for each chromosome

tool:	GenomeAnalysisTK.jar -T RealignerTargetCreator?
input:	genome.chr.fa, dbsnpXYz.chr.rod, indelsXYZ.vcf
output:	genome.chr.realign.intervals
doc:	​http://www.broadinstitute.org/gsa/wiki/index.php/Local_realignment_around_indels#Running_the_Indel_Realigner_only_at_known_sites

Workflow 2: Alignment per Lane, per Chr

This workflow aligns reads per lane and chromosome, including:

• re-alignment to prevend false SNP calls caused by indels (using known indels)
• markduplicates to prevend false coverage caused by PCR errors (per library = lane)
• base quality recalibration to correct for false low scores caused by true variation

Workflow Inputs:

• lane.1.fq.gz - raw reads for lane, pair end 1
• lane.2.fq.gz - raw reads for lane, pair end 2
• genome.chr.fasta - reference genome split on chromosome
• genome.chr.realign.intervals - targets for realignment per chromosome
• genome.chr.dbsnpXYZ.rod - known snp variants, here from dpbsnp
• genome.chr.indelsXYZ.vcf - known indels from, here from 1KG

Workflow ouputs:

• lane.chr.1.sai - alignment index for first pair
• lane.chr.2.sai - alignment index for second pair
• lane.chr.sam - alignment map for
• lane.chr.bam - alignment map in binary format
• lane.chr.sorted.bam - sorted alignment map
• lane.chr.sorted.bai - sorted alignment index
• lane.chr.dedup.bam - marked duplicate PCR elements
• lane.chr.dedup.metrics - metrics describing deduplication
• lane.chr.realigned.bam - realigned based on known indels
• lane.chr.matefixed.bam - fixed the mate pair ends
• lane.chr.covariate_table.csv - table of countcovariates output for recalibration
• lane.chr.recal.bam - alignment map with recalibrated quality scores

align

Align each end of paired end.

tool:	bwa-align
input:	chr.fasta, lane.1.fq.gz, lane.2.fq.gz
output:	lane.chr.1.sai, lane.chr.2.sai
docs:	​http://bio-bwa.sourceforge.net/bwa.shtml

align-pe

Align the pairs as one

tool:	bwa sampe
inputs:	chr.fasta lane.1.fq.gz lane.2.fq.gz lane.chr.1.sai lane.chr.2.sai
outputs:	lane.chr.sam
docs:	​http://bio-bwa.sourceforge.net/bwa.shtml

sam-to-bam

Convert sam to bam

tool:	samtools view
inputs:	lane.chr.sam
outputs:	lane.chr.bam
docs:	​http://samtools.sourceforge.net/samtools.shtml

(Question: can this not index and sort?)

sam-sort

Sort bam file on coordinate

tool:	samtools sort
inputs:	lane.chr.bam
outputs:	lane.chr.sorted.bam
docs:	​http://samtools.sourceforge.net/samtools.shtml

sam-index

Index bam file for quicker access

tool:	samtools index
inputs:	lane.chr.sorted.bam
outputs:	lane.chr.sorted.bai
docs:	​http://samtools.sourceforge.net/samtools.shtml

MarkDuplicates

Mark duplicate PCR fragments to be filtered in analysis

tool:	MarkDuplicates?.jar
inputs:	lane.chr.sorted.bam
outputs:	lane.chr.dedup.bam lane.chr.dedup.metrics
docs:	​http://picard.sourceforge.net/command-line-overview.shtml#MarkDuplicates

IndelRealigner-KnownsOnly

Improve the alignment using known indel information (will reduce false SNP calls)

tool:	GenomeAnalysisTK.jar -T IndelRealigner?
inputs:	lane.chr.dedup.bam genome.chr.realign.intervals genome.chr.dbsnpXYZ.rod genome.chr.indelsXYZ.vcf
outputs:	lane.chr.realigned.bam
docs	​http://www.broadinstitute.org/gsa/wiki/index.php/Local_realignment_around_indels#Running_the_Indel_Realigner_only_at_known_sites

FixMateInformation

Fix the paired end information as consequence of the realignment.

tool:	FixMateInformation?.jar
inputs:	lane.chr.realigned.bam
outputs:	lane.chr.matefixed.bam
docs:	​http://picard.sourceforge.net/command-line-overview.shtml#FixMateInformation,

​http://www.broadinstitute.org/gsa/wiki/index.php/Local_realignment_around_indels#Fixing_Mate_Pairs

CountCovariates

Count covariants, such as machine cycle and bp position, to be used as basis for quality recalibration. Optionally: plot the results to pdf using AnalyzeCovariates?

tool:	GenomeAnalysisTK.jar -T CountCovariates?, AnalyzeCovariates?.jar
inputs:	lane.chr.matefixed.bam genome.chr.dbsnpXYZ.rod
outputs:	lane.chr.covariate_table.csv
docs:	​http://www.broadinstitute.org/gsa/wiki/index.php/Base_quality_score_recalibration#CountCovariates

​http://www.broadinstitute.org/gsa/wiki/index.php/Base_quality_score_recalibration#AnalyzeCovariates.jar

TableRecalibration

Recalibrate quality scores based on the covariate table

tool:	GenomeAnalysisTK.jar -T TableRecalibration?
inputs:	lane.chr.matefixed.bam lanec.chr.recal_table.csv chr.fasta
outputs:	lane.chr.recal.bam
docs:	​http://www.broadinstitute.org/gsa/wiki/index.php/Base_quality_score_recalibration#TableRecalibration

Repeat: sam-sort, sam-index, countcovariates

See steps above for commands and docs.

inputs:	lane.chr.recal.bam
outputs:	lane.chr.recal.sorted.bam, lane.chr.recal.sorted.bam.bai, lane.chr.recal.covariate_table.csv

Discussion:

wy do we need to sort and index after recalibration? does it mess up the order of things?

Workflow 3: sample level variant calling

This workflow will call variants for the samples including:

• sample level recalibration
• sample level realignment

N.B. no sample level MarkDuplicates? is needed as lanes == libraries.

Workflow inputs:

• lane.chr.recal.sorted.bam - for all sample lanes: dedupped, recalibrated, realigned, sorted and indexed bams (3)
• sample.chip.vcf - genotypes called from genotype chip

Reference:

• genome.chr.fasta - reference genome split on chromosome
• genome.chr.realign.intervals - targets for realignment per chromosome
• genome.chr.dbsnpXYZ.rod - known snp variants, here from dpbsnp
• genome.chr.indelsXYZ.vcf - known indels from, here from 1KG

Workflow outputs:

• sample.chr.bam - merged bam files per sample
• sample.chr.realign.interval - realignment target intervals
• sample.chr.realigned.bam - realigned
• sample.chr.matesfixed.bam - fixed pairs in realignment
• sample.chr.indels.vcf - raw indels called
• sample.chr.indels.bed - raw indels annotations
• sample.chr.indels.txt - output from the indel calling
• sample.chr.indels.filtered.bed - indels filtered
• sample.chr.snps.vcf - raw snps called
• sample.chr.snps.filtered.vcf - snps filtered

merge-lanes

Merge lanes into one sample bam

tool:	sam merge
inputs:	lane.chr.recal.sorted.bam
outputs:	sample.chr.bam
docs:	​http://samtools.sourceforge.net/samtools.shtml

RealignerTargetCreator

Create realignment targets based on the data (so not only knowns)

tool:	GenomeAnalysisTK.jar -T RealignerTargetCreator?
inputs:	sample.chr.bam genome.chr.fa dbsnpXYz.chr.rod indelsXYZ.vcf
outputs:	sample.chr.realign.intervals
doc:	​http://www.broadinstitute.org/gsa/wiki/index.php/Local_realignment_around_indels#Creating_Intervals

IndelRealigner

Realign based on realignment targets in previous step

tool:	GenomeAnalysisTK.jar -T IndelRealigner?
inputs:	sample.chr.bam genome.chr.realign.intervals genome.chr.dbsnpXYZ.rod genome.chr.indelsXYZ.vcf
outputs:	sample.chr.realigned.bam
doc:	​http://www.broadinstitute.org/gsa/wiki/index.php/Local_realignment_around_indels#Realigning

FixMateInformation

See description in workflow2, now applied to sample

inputs:	sample.chr.realigned.bam
ouputs:	sample.chr.matesfixed.bam

IndelGenotyperV2

Call indels

tool:	GenomeAnalysisTK.jar -T IndelGenotyperV2
inputs:	sample.chr.matesfixed.bam genome.chr.fa
outputs:	sample.chr.indels.vcf sample.chr.indels.bed sample.chr.indels.txt
doc:	​http://www.broadinstitute.org/gsa/wiki/index.php/Indel_Genotyper_V2.0

​http://www.broadinstitute.org/gsa/wiki/index.php/Firehose_Parameters#SampleIndelGenotyper

filterSingleSampleCalls

Filter indels

tool:	filterSingleSampleCalls.pl
inputs:	sample.chr.indels.bed
outputs:	sample.chr.indels.filtered.bed
doc:	​http://www.broadinstitute.org/gsa/wiki/index.php/Firehose_Parameters#SampleIndelGenotyper

UnifiedGenotyper

Call SNPs

tool:	GenomeAnalysisTK.jar -T UnifiedGenotyper?
inputs:	sample.chr.matesfixed genome.chr.fa dbsnpXYz.chr.rod
outputs:	sample.chr.snps.vcf
doc:	​http://www.broadinstitute.org/gsa/wiki/index.php/Firehose_Parameters#SetUnifiedGenotypertoEval

​http://www.broadinstitute.org/gsa/wiki/index.php/Unified_genotyper

makeIndelMask

Make indel mask

tool:	makeIndelMask.py
inputs:	sample.chr.indels.bed
outputs:	sample.chr.indels.mask.bed
doc:	​http://www.broadinstitute.org/gsa/wiki/index.php/Indel_Genotyper_V2.0#Creating_a_indel_mask_file

VariantFiltration

Filter variants to get the best calls possible

tool:	GenomeAnalysisTK.jar -T VariantFiltration?
inputs:	sample.chr.snps.vcf genome.chr.fa dbsnpXYz.chr.rod
outputs:	sample.chr.snps.filtered.vcf
doc:	​http://www.broadinstitute.org/gsa/wiki/index.php/Best_Practice_Variant_Detection_with_the_GATK_v2#Integrating_analyses:_getting_the_best_call_set_possible

MergeVcfs

ChipVcf

Produce vcf for the chips

VariantEval

Create summary information on the variations called for evaluation. Run per sample.snps.filtered.vcf against chip.

tool:	GenomeAnalysisTK.jar -T VariantEval?
inputs:	sample.snps.vcf sample.chip.vcf genome.chr.fa dbsnpXYz.chr.rod
outputs:	sample.snps.eval
doc:	​http://www.broadinstitute.org/gsa/wiki/index.php/VariantEval

Discussion:

Do we call SNPs based on the filtered indels or the raw indels? Should we realign AGAIN after merge of lanes? BAQ? MINDEL/PINDEL?