Changes between Version 5 and Version 6 of SOPs/variant_calling

Timestamp:

12/11/13 16:14:22 (11 years ago)

Author:

gbell

Comment:

--

SOPs/variant_calling

@@ -5 +5 @@
 * mapping short reads
 * calling raw variants
-* adding filters (really more like 'tagging') and some annotations to variants
-* annotating effect(s) of variants on genes
+* adding filters (really more like 'tagging' to identify raw variants that are really variants and not technical errors) and some annotations to variants
+* annotating effect(s) of variants on genes (like if they change protein sequence)
 
 == Map short reads ==
@@ -38 +38 @@
 }}}
 
-Get uniquely mapping reads (?)
+Get uniquely mapping reads (Is this recommended or not?)
 {{{
 samtools view -h A_reads.bt2.bam | grep -v XS:i: | samtools view -bS - > A_reads.bt2.sorted_unique.bam
 }}}
 
-Remove duplicated reads assumed to be PCR artifacts (?)
+Remove duplicated reads assumed to be PCR artifacts.  This should be done even if duplication rate is low.
 {{{
 # paired-end reads
@@ -53 +53 @@
 == Call raw variants with mpileup+bcftools ==
 
-Call variants (one sample vs. reference) with samtools' mpileup+bcftools (see the [http://samtools.sourceforge.net/mpileup.shtml| samtools' variant calling page] for more details)
+Call variants (one sample vs. reference) with samtools' mpileup+bcftools (see the [http://samtools.sourceforge.net/mpileup.shtml| samtools' variant calling page] for more details).
+In our experience, "-B" is needed to disable BAQ computation, which would otherwise remove many obvious variants.
 {{{
-samtools mpileup -d100000 -uf /nfs/genomes/sgd_2010/bwa/sacCer3.fa A_reads.bt2.sorted_unique.bam | bcftools view -bvcg - >| A_reads.bt2.sorted_unique.raw.bcf
+samtools mpileup -d100000 -Buf /nfs/genomes/sgd_2010/bwa/sacCer3.fa A_reads.bt2.sorted_unique.bam | bcftools view -bvcg - >| A_reads.bt2.sorted_unique.raw.bcf
 }}}
 Call variants (multiple sample vs. reference) using a set of BAM files
 {{{
-samtools mpileup -d100000 -uf /nfs/genomes/sgd_2010/bwa/sacCer3.fa *_reads.bt2.sorted_unique.bam | bcftools view -bvcg - >| ALL_reads.bt2.sorted_unique.raw.bcf
+samtools mpileup -d100000 -Buf /nfs/genomes/sgd_2010/bwa/sacCer3.fa *_reads.bt2.sorted_unique.bam | bcftools view -bvcg - >| ALL_reads.bt2.sorted_unique.raw.bcf
 }}}
 
@@ -66 +67 @@
 This step uses vcf-annotate from the [http://vcftools.sourceforge.net/docs.html VCFtools suite]
 
-Annotate variants by adding tags ("filters" but all variants are kept) to each variant, using all default filters
+Annotate variants by adding tags ("filters" but all variants are kept) to each variant, using all default filters.
 {{{
 bcftools view -L -vcg A_reads.bt2.sorted_unique.raw.bcf | vcf-annotate -f + > A_reads.bt2.sorted_unique.withTags.bcf