= 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 ||
||command:
[[IncludeSource(ngs_pipelines/templates/ngs/template-paired-end_step_0_1.ftl) ||

== 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?