Changes between Initial Version and Version 1 of SnpCallingPipeline/AlignmentAndCleaning

Timestamp:

Oct 18, 2010 4:44:50 AM (14 years ago)

Author:

Morris Swertz

Comment:

--

SnpCallingPipeline/AlignmentAndCleaning

@@ +1 @@
+= Workflow 2: Alignment per Lane, per Chr =
+[[TOC()]]
+
+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 [[BR]] lane.1.fq.gz [[BR]] lane.2.fq.gz [[BR]] lane.chr.1.sai [[BR]] 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 [[BR]] 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 [[BR]] genome.chr.realign.intervals [[BR]] genome.chr.dbsnpXYZ.rod [[BR]] 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 [[BR]] genome.chr.dbsnpXYZ.rod ||
+||outputs: ||lane.chr.covariate_table.csv ||
+||docs:    ||http://www.broadinstitute.org/gsa/wiki/index.php/Base_quality_score_recalibration#CountCovariates [[BR]]
+
+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 [[BR]]lanec.chr.recal_table.csv [[BR]]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?