Changes between Initial Version and Version 1 of SOPs/callingSNPs

Timestamp:

01/23/13 16:49:43 (12 years ago)

Author:

trac

Comment:

--

SOPs/callingSNPs

@@ +1 @@
+== SNP Calling ==
+=== mpileup (SAMtools) ===
+  * Create bcf file (binary version of vcf) from alignment file (bam)
+
+
+{{{
+  #Use Q to set base quality threshold
+  samtools mpileup -Q 25 -ugf <reference.fasta> <file.bam> | bcftools view -bvcg - > accepted_hits.raw.bcf
+}}}
+
+
+  * Convert bcf to vcf (vairant call format) and filter, if needed
+
+  
+{{{
+#filter using varFilter (from vcfutils)
+  #use Q to set mapping quality
+  #use d for minimum read depth
+  bcftools view accepted_hits.raw.bcf | vcfutils.pl varFilter -d 5 -Q 20 >| accepted_hits.raw.vcf
+}}}
+
+
+
+== Filtering SNPs ==
+
+  * Remove, or retrieve, known SNPs
+
+
+{{{
+  #To remove known SNPs, use intersectBed (bedtools).  Known SNPs can be downloaded from Ensembl or NCBI/UCSC (eg. dbSNP)
+  intersectBed -a accepted_hits.raw.filtered.vcf -b Mus_musculus.NCBIM37.60.bed -wo > filteredSNPs.vcf
+}}}
+
+  
+== Annotating SNPs ==
+    * Determining the effect of the SNP
+
+=== snpEff ===
+ 
+{{{
+ #Use appropriate i) organism  and ii) annotation (eg. UCSC, RefSeq, Ensembl)
+  #Mouse: mm37 (UCSC/RefSeq), mm37.61 (Ensembl)
+  #Human: hg37 (UCSC/RefSeq), hg37.61 (Ensembl)
+  #Output is created in several files: an html summary file and text files with detailed information 
+  snpEff -vcf4 mm37.61 accepted_hits.raw.filtered.vcf > accepted_hits.raw.filtered.snpEff
+
+}}}