How to generate vcf using samtools Version: 0.1.19-96b5f2294a?

Question

[lakhujanivijay]

I am new to NGS and I am stuck at a point where I need to generate .vcf file using a BAM file and a genome. I am on Ubuntu 14.0. I have samtools v0.1.19-96b5f2294a

Here is the output when I type in samtools mpileup and hit enter

 samtools mpileup

 Usage: samtools mpileup [options] in1.bam [in2.bam [...]]

Input options:

   -6           assume the quality is in the Illumina-1.3+ encoding
   -A           count anomalous read pairs
   -B           disable BAQ computation
   -b FILE      list of input BAM filenames, one per line [null]
   -C INT       parameter for adjusting mapQ; 0 to disable [0]
   -d INT       max per-BAM depth to avoid excessive memory usage [250]
   -E           recalculate extended BAQ on the fly thus ignoring existing BQs
   -f FILE      faidx indexed reference sequence file [null]
   -G FILE      exclude read groups listed in FILE [null]
   -l FILE      list of positions (chr pos) or regions (BED) [null]
   -M INT       cap mapping quality at INT [60]
   -r STR       region in which pileup is generated [null]
   -R           ignore RG tags
   -q INT       skip alignments with mapQ smaller than INT [0]
   -Q INT       skip bases with baseQ/BAQ smaller than INT [13]
   --rf INT     required flags: skip reads with mask bits unset []
   --ff INT     filter flags: skip reads with mask bits set []

  Output options:

   -D           output per-sample DP in BCF (require -g/-u)
   -g           generate BCF output (genotype likelihoods)
   -O           output base positions on reads (disabled by -g/-u)
   -s           output mapping quality (disabled by -g/-u)
   -S           output per-sample strand bias P-value in BCF (require -g/-u)
   -u           generate uncompress BCF output

  SNP/INDEL genotype likelihoods options (effective with `-g' or `-u'):

   -e INT       Phred-scaled gap extension seq error probability [20]
   -F FLOAT     minimum fraction of gapped reads for candidates [0.002]
   -h INT       coefficient for homopolymer errors [100]
   -I           do not perform indel calling
   -L INT       max per-sample depth for INDEL calling [250]
   -m INT       minimum gapped reads for indel candidates [1]
   -o INT       Phred-scaled gap open sequencing error probability [40]
   -p           apply -m and -F per-sample to increase sensitivity
   -P STR       comma separated list of platforms for indels [all]

Notes: Assuming diploid individuals.

I am giving following command:

samtools mpileup –f wu_0.v7.fas –uv out.full.sorted.bam > out.full.mpileup.vcf

I get below error:

mpileup: invalid option -- 'v'
[mpileup] 1 samples in 1 input files
<mpileup> Set max per-file depth to 8000

Please help

Answer 1

[Devon Ryan]

As you can see, there is no "-v" option in that version. You need to pipe the bcf file to bcftools.

[lakhujanivijay]

Thanks Devon,

But sorry I am very naive user. Can you help me with the command set?

here are some details:

genome - wu_0.v7.fas

BAM file - out.full.sorted.bam (i sorted it using samtools)

Please help

[Devon Ryan]

Read the output of "bcftools view". Note that I really can't recommend using such outdated versions of samtools and bcftools through.