Comprehensive Samtools user guide?

[wdemos]

I'm a first time samtools user. I am using the mpileup command for somatic mutation calling between a pair of samples. I generate an output file: var.bcf, but I don't know how to open/extract the data from the file. Is there a comprehensive user guide somewhere with examples/tutorials how to use this tool? I've been to http://samtools.sourceforge.net/samtools.shtml but it is no where near detailed enough for my level of experience with this tool. Thank you in advance for any insight.

[swbarnes2]

The quick answer...

bcftools view var.bcf > var.vcf

That's a plain text file

[wdemos]

Thank you. I did figure out how to view the file, the interpretation is the issue. It looks like a lot oh headers but no data:
##fileformat=VCFv4.1
##INFO=<ID=DP,Number=1,Type=Integer,Description="Raw read depth">
##INFO=<ID=DP4,Number=4,Type=Integer,Description="# high-quality ref-forward bases, ref-reverse, alt-forward and alt-reverse bases">
##INFO=<ID=MQ,Number=1,Type=Integer,Description="Root-mean-square mapping quality of covering reads">
##INFO=<ID=FQ,Number=1,Type=Float,Description="Phred probability of all samples being the same">
##INFO=<ID=AF1,Number=1,Type=Float,Description="Max-likelihood estimate of the first ALT allele frequency (assuming HWE)">
##INFO=<ID=AC1,Number=1,Type=Float,Description="Max-likelihood estimate of the first ALT allele count (no HWE assumption)">
##INFO=<ID=G3,Number=3,Type=Float,Description="ML estimate of genotype frequencies">
##INFO=<ID=HWE,Number=1,Type=Float,Description="Chi^2 based HWE test P-value based on G3">
##INFO=<ID=CLR,Number=1,Type=Integer,Description="Log ratio of genotype likelihoods with and without the constraint">
##INFO=<ID=UGT,Number=1,Type=String,Description="The most probable unconstrained genotype configuration in the trio">
##INFO=<ID=CGT,Number=1,Type=String,Description="The most probable constrained genotype configuration in the trio">
##INFO=<ID=PV4,Number=4,Type=Float,Description="P-values for strand bias, baseQ bias, mapQ bias and tail distance bias">
##INFO=<ID=INDEL,Number=0,Type=Flag,Description="Indicates that the variant is an INDEL.">
##INFO=<ID=PC2,Number=2,Type=Integer,Description="Phred probability of the nonRef allele frequency in group1 samples being larger (,smaller) than in group2.">
##INFO=<ID=PCHI2,Number=1,Type=Float,Description="Posterior weighted chi^2 P-value for testing the association between group1 and group2 samples.">
##INFO=<ID=QCHI2,Number=1,Type=Integer,Description="Phred scaled PCHI2.">
##INFO=<ID=PR,Number=1,Type=Integer,Description="# permutations yielding a smaller PCHI2.">
##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype">
##FORMAT=<ID=GQ,Number=1,Type=Integer,Description="Genotype Quality">
##FORMAT=<ID=GL,Number=3,Type=Float,Description="Likelihoods for RR,RA,AA genotypes (R=ref,A=alt)">
##FORMAT=<ID=DP,Number=1,Type=Integer,Description="# high-quality bases">
##FORMAT=<ID=SP,Number=1,Type=Integer,Description="Phred-scaled strand bias P-value">
##FORMAT=<ID=PL,Number=-1,Type=Integer,Description="List of Phred-scaled genotype likelihoods, number of values is (#ALT+1)*(#ALT+2)/2">
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT

Not sure what I did wrong. The mplieup command I used was:
samtools mpileup -DSuf ref.fa aln.bam | bcftools view -bvcgT pair - > var.bcf (inclded my filepaths/names of course)

[swbarnes2]

At first glance, you have no SNPs.

Second, for some reason all the reads that show varaints have been filtered away.

Try using -A or -B in your mpileup command.

Try removing the '-T pair' from bcftools

[wdemos]

thank you I will gie your suggestions a go.

[wdemos]

I didn't have the files aligned correctly to the alignment file causing the error. Thank you for the help.