<p>(cross posted on seqanswers: <a href='http://seqanswers.com/forums/showthread.php?t=12542'>http://seqanswers.com/forums/showthread.php?t=12542</a> )</p>

<p>Hi all,</p>

<p>I know, by capillary sequencing, that one of my samples contains a mutation at position:<code>120458492</code>.</p>

<p>Some reads were aligned with <strong>bwa</strong> and I can clearly see my mutation using <strong>samtools tview</strong>.</p>

<pre><code>       120458491 120458501
CCTGCTCTGGGGAGCTATGCCAGGATGGGTGCC
........R........................
.....C..A..................C.....
........C. ......................
............  ...................
........A.....    ...............
,,,,,,,,a,,,,,,,,,,,,,,,,  ......
,,,,,,,,a,,,,,,,,,,,,,,,,,,,    .
........A........................
........A........................
.............................C...
........A........................
.................................
........A........................
.................................
.................................
........A........................
......................C..........
........A........................
,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,
........A........................
.................................
 ................................
  ...............................
   .....A........................
      ,,,,,,,,,gg,,,,,,,,,,,t,,,,
       .A........................
        .........................
</code></pre>

<p>however, we I call the mutations using <strong>samtools mpileup</strong> </p>

<pre><code>${SAMTOOLS}/samtools mpileup -uf  ${HG19} sample.bam |\
  ${SAMTOOLS}/bcftools/bcftools view  -bvcg - &gt; snps.bcf
${SAMTOOLS}/bcftools/bcftools view  snps.bcf | gzip --best &gt; snps.vcf.gz
</code></pre>

<p>But I can't see the mutation in the VCF.</p>

<p>How can I know why mpileup (or bcftools ?)  skipped this mutation ?</p>

<p>Thanks !</p>

<p>Pierre</p>

<p>EDIT:</p>

<p>...and here is the output of <strong>pileup</strong> (not mpileup):</p>

<pre><code>(...)
chr1    120458492    G    26    C.AaaAA.A.A..A.A,A...A,AA^].    JddfhQacfhhgggahehhhhaB[hg
(...)
</code></pre>

<p>Here is the solution , suggested by <a href='http://seqanswers.com/forums/member.php?u=480'>swbarnes2</a> on seqanswers:</p>

<blockquote>
  <p>Try redoing mpileup with -Buf. That's
  worked for me. Like you, I observed
  that my sanger-verified mutation
  looked fine in pileup, but when I
  looked at it in the mpileup run
  without -B, the Baq calculations
  destroyed the quality scores of that
  locus, so it wouldn't call a SNP.
  Running it with -B should make your
  mpileup look like your pileup at that
  locus, and then the SNP will have high
  enough quality to be counted.</p>
</blockquote>

<pre><code>$ gunzip -c snps.vcf.gz | grep chr1 | grep "120458492"
chr1    120458492    .    G    A    99    .    DP=26;AF1=0.5;CI95=0.5,0.5;DP4=10,2,12,2;MQ=60;PV4=1,0.37,1,1    PL:GT:GQ    255,0,255:0/1:99
</code></pre>
