hands-on-session-2.1-bcftools

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Hands-on Session 3: bcftools Xian Mallory
bcftools • A program for variant calling and manipulating files in the Variant Call Format (VCF) and its binary counterpart BCF . • All commands work transparently with both VCFs (uncompressed) and BCFs ( BGZF-compressed ). • Manual for bcftools https://samtools.github.io/bcftools/bcftools.html • Examples of how to use bcftools can be found in http://samtools.github.io/bcftools/howtos/index.html
Installation • This webpage shows how to install the software: http://samtools.github.io/bcftools/howtos/install.html • RCC already installed bcftools. • Use `module load gnu` to load it if `which bcftools` does not locate the bcftools that you are using.
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Usage of bcftools • Extracting information from bcftools ( https://samtools.github.io/bcftools/howtos/query.html ) • bcftools query • Filtering ( https://samtools.github.io/bcftools/howtos/filtering.html ) • bcftools query –e’FILTER=“.”’ • Viewing a vcf file ( https://samtools.github.io/bcftools/bcftools.html#view ) • bcftools view • Intersection of two vcf files ( https://samtools.github.io/bcftools/bcftools.html#isec ) • bcftools isec • Merging two or more vcf files ( https://samtools.github.io/bcftools/bcftools.html#merge ) • bcftools merge
Data preparation (1) • Login to RCC, and run the following command to copy the data to your home directory. cp -r /gpfs/research/scratch/xfan2/bcftoolsSession ./ • In your folder (~/bcftoolsSession/), you can find a file named “ chr22.vcf.gz ”, which is a vcf files containing variants from hundreds of samples (all healthy genomes) on chr22. • “chr22.vcf.gz” has a corresponding index file, named “chr22.vcf.gz.csi”. • In the next few slides, we will try to query and view “chr22.vcf.gz” from a variety of perspectives.
bcftools query for extracting information • Extracting the samples ( -l ) bcftools query -l chr22.vcf.gz | less • How many samples are in this file?
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bcftools query for extracting information (con’d) • Extracting the positions ( -f : specifying format) bcftools query -f '%POS\n' chr22.vcf.gz | less • Extracting the positions with a certain format bcftools query -f 'pos=%POS\n' chr22.vcf.gz | less • Extracting the chromosome, positions, reference and alternative base(s) bcftools query -f '%CHROM %POS %REF %ALT\n' chr22.vcf.gz | less
bcftools query for filtering • Only output those lines whose reference value is not “A” ( -e , --exclude) bcftools query -e'REF="A"' -f'%CHROM %POS %REF\n' chr22.vcf.gz | less
bcftools query for filtering (con’d) • Only output those lines whose QUAL > 20 ( -i , --include) bcftools query -i'QUAL>20' -f'%CHROM %POS %QUAL\n' chr22.vcf.gz | less
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bcftools view for outputting a certain region • To use bcftools view, we need to index the vcf file first. Notice that your folder already contains the index file (.csi) as it takes time to run this command. • An alternative way to create an index file is “tabix chr22.vcf.gz”, which would create a .tbi file. bcftools index chr22.vcf.gz • “bcftools view” can select a certain region like “samtools view” does. bcftools view -H chr22.vcf.gz 22:16000000-17000000 | less • Use –S in “less” command to switch the view to one row per line. • -H here means no header.
bcftools view for viewing header only (-h) bcftools view -h chr22.vcf.gz | less • If no –H and –h , header will be printed together with the data section.
bcftools view for viewing selected sample(s) only (-s) bcftools view -H -s NA12878 chr22.vcf.gz 22:16000000-17000000 | less
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Now let’s switch to a different data set for testing ` bcftools isec ` and ` bcftools merge `.
Data preparation (2) • In your folder (~/ bcftoolsSession/), you will find the vcf files for a family, father (HG003) , mother (HG004) and son (HG002) , for the variants that come only from chr22. This is a dataset publicly available and from Ashkenazim trio who are Jews. • For each sample, you will have *.chr22.vcf.gz (compressed vcf file) and *.chr22.vcf.gz.csi (index file), in which * could be father, mother or son. • Now let’s make a comparison between father and son first.
bcftools isec for comparing two vcf files (con’d) Given two vcf files, the following command can create the intersection and the complements between the variants from the two given vcf files. bcftools isec -p comp_ashtrio_father_son son.chr22.vcf.gz father.chr22.vcf.gz Go into the folder “comp_ashtrio_father_son” and examine the four files just created.
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bcftools isec for comparing two vcf files (con’d) In folder “comp_ashtrio_father_son”, there are four files, • 0000.vcf variants private to son • 0001.vcf variants private to father • 0002.vcf variants from son but shared by both son and father • 0003.vcf variants from father but shared by both son and father In 0002.vcf, the column after FORMAT shows the data from son only. In 0003.vcf, the column after FORMAT shows the data from father only. Note : the entries from father and son are not merged yet in the files created by `bcftools isec`.
bcftools merge for merging two vcf files • Now we do the merge. bcftools merge -O z -o father.son.chr22.vcf.gz father.chr22.vcf.gz son.chr22.vcf.gz • Here “-o father.son.chr22.vcf.gz” specifies that the output file is “father.son.chr22.vcf.gz”. • “–O z” specifies that the output file (father.son.chr22.vcf.gz) should be in a compressed vcf format. • In the merged file, check the genotypes for chr22:15271151, the one that was reported by both 0002.vcf and 0003.vcf by `bcftools isec` (shared by father and son). Also check this position for father.chr22.vcf.gz and son.chr22.vcf.gz, respectively.
bcftools merge for merging multiple vcf files (con’d) • Merge vcf files from father, mother and son, so that there will be extra three columns after the FORMAT column. bcftools merge -O z -o trio.chr22.vcf.gz father.chr22.vcf.gz mother.chr22.vcf.gz son.chr22.vcf.gz • Now in the merged file, check the genotypes for chr22:15271151 again. You can see that the second column after the FORMAT column, (corresponding to mother’s) does not report anything. • Is this situation biologically possible? • What does this indicate?
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