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.

[[IncludeSource?(ngs_pipelines/templates/ngs/template-paired-end_step_0_1.ftl)

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
command:

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?