Difference between revisions of "CallSNPs.py"

From wiki
Jump to: navigation, search
Line 9: Line 9:
  
 
'''mpileup''' collects summary information from the input BAMs and then computes and stores the likelihood of data given each possible genotype. '''bcftools''' applies to prior and then calls the variants. Can be quite raw so often a filter is needed after this.
 
'''mpileup''' collects summary information from the input BAMs and then computes and stores the likelihood of data given each possible genotype. '''bcftools''' applies to prior and then calls the variants. Can be quite raw so often a filter is needed after this.
 
  
 
==2. Filter variants through a maximum read depth ==
 
==2. Filter variants through a maximum read depth ==
 
  bcftools view ''bcf_file'' | vcfutils.pl varFilter -D''NUM''
 
  bcftools view ''bcf_file'' | vcfutils.pl varFilter -D''NUM''
'''bcftools view''' output the variants in VCF format and '''vcfutils.pl''' perl script , which is part of samtools platform, filters for variants via the ''-D'' option that sets the maximum  value.
+
'''bcftools view''' outputs the variants in VCF format and '''vcfutils.pl''' perl script , which is part of samtools platform, filters for variants on their maximum value via the ''-D'' option.
  
 
== 3. Compress vcf files==
 
== 3. Compress vcf files==
Not strictly part of the sequential run of the pipeline, but at this point the vcf files are compressed with '''samtool''''s bgzip and then indexed with '''tabix''' (also from '''samtools''', a generic tab-file delimited file indexer).
+
Not strictly part of the sequential run of the pipeline, but at this point the vcf files are compressed with '''samtools'''' bgzip and then indexed with '''tabix''' (also from '''samtools''', a generic tab-file delimited file indexer).
 
  bgzip -c ''vcf_file'' > ''vcf_file.gz''
 
  bgzip -c ''vcf_file'' > ''vcf_file.gz''
 
  tabix -p vcf ''vcf_file.gz''
 
  tabix -p vcf ''vcf_file.gz''
Line 24: Line 23:
 
  bedtools genomecov -bga -ibam ''input_bam_file'' | awk -v t=''NUM'' "{if($4>=t)print}" | bedtools merge -i - > ''output_bed_file''
 
  bedtools genomecov -bga -ibam ''input_bam_file'' | awk -v t=''NUM'' "{if($4>=t)print}" | bedtools merge -i - > ''output_bed_file''
  
'''bedtools genomecov''' outputs the number of aligned short-read bases in the genome in a format (enabled by the '''-bga''' option) in the fourth column. This is picked up by the '''awk''' program which ignores all those less than '''NUM'''. If '''NUM''' is set to zero, the entire variant callset will be returned in raw form becuase nothing will be filtered and the '''-bga''' (with '''-a''' presumably standing for ''all'')  includes areas with zero coverage (this sounds uninteresting but it could be useful to have, so it's often kept). The final merge, as well as merging the filtered output, also makes sure that split and overlapping features are rolled into one.
+
'''bedtools genomecov''' outputs the number of aligned short-read bases in the genome in a format (enabled by the '''-bga''' option) in the fourth column. This is picked up by the '''awk''' program which ignores all those less than '''NUM'''. If '''NUM''' is set to zero, the entire variant callset will be returned in raw form because nothing will be filtered and the '''-bga''' (with '''-a''' presumably standing for ''all'')  will include areas with zero coverage (this sounds uninteresting but it could be useful to have, so it's often kept). The final merge, as well as merging the filtered output, also makes sure that split and overlapping features are rolled into one. Note that at this point our variant format VCF turns into a BED file.
 
 
 
 
  
 
== 5. intersection op ==
 
== 5. intersection op ==
 
  bedtools intersect -a intersect_15.bed.temp -b ../altsortedbam/ERR128717_S.15.bed > intersect_15.bed.temp_2
 
  bedtools intersect -a intersect_15.bed.temp -b ../altsortedbam/ERR128717_S.15.bed > intersect_15.bed.temp_2

Revision as of 11:24, 3 April 2016

The call SNPS.py script is part of "script_tools" modules.

It requires an alignment in the shape of the base reference sequence and the bam files of the short reads aligned to that sequence (run previously, maybe with bwa or one of the bowtie programs).

Stages

1. Calculate likelihoods of each possible variant, and apply prior and call variants

samtool mpileup -E -d NUM -DSugB -Q 1 -f ref_file input_bam_file | bcftools view -cgbu - > output_bcf_file

mpileup collects summary information from the input BAMs and then computes and stores the likelihood of data given each possible genotype. bcftools applies to prior and then calls the variants. Can be quite raw so often a filter is needed after this.

2. Filter variants through a maximum read depth

bcftools view bcf_file | vcfutils.pl varFilter -DNUM

bcftools view outputs the variants in VCF format and vcfutils.pl perl script , which is part of samtools platform, filters for variants on their maximum value via the -D option.

3. Compress vcf files

Not strictly part of the sequential run of the pipeline, but at this point the vcf files are compressed with samtools' bgzip and then indexed with tabix (also from samtools, a generic tab-file delimited file indexer).

bgzip -c vcf_file > vcf_file.gz
tabix -p vcf vcf_file.gz

The latter command creates an extra vcf_file.gz.tbi file in the directory.

4. Extract features with minimum coverage

bedtools genomecov -bga -ibam input_bam_file | awk -v t=NUM "{if($4>=t)print}" | bedtools merge -i - > output_bed_file

bedtools genomecov outputs the number of aligned short-read bases in the genome in a format (enabled by the -bga option) in the fourth column. This is picked up by the awk program which ignores all those less than NUM. If NUM is set to zero, the entire variant callset will be returned in raw form because nothing will be filtered and the -bga (with -a presumably standing for all) will include areas with zero coverage (this sounds uninteresting but it could be useful to have, so it's often kept). The final merge, as well as merging the filtered output, also makes sure that split and overlapping features are rolled into one. Note that at this point our variant format VCF turns into a BED file.

5. intersection op

bedtools intersect -a intersect_15.bed.temp -b ../altsortedbam/ERR128717_S.15.bed > intersect_15.bed.temp_2