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  • problems understanding pileup format

    Hello

    I have been given some data in pileup format. Whilst it is beyond the scope of my work to go into the details of how the reads/alignments etc are generated I think it would be remiss of me not to try and understand a bit more about what is going on.

    I have read the SAM tools manual and explanation of the pileup format but still don't understand a few things. Here is an example taken from the manual


    seq1 272 T 24 ,.$.....,,.,.,...,,,.,..^+. <<<+;<<<<<<<<<<<=<;<;7<&
    seq1 273 T 23 ,.....,,.,.,...,,,.,..A <<<;<<<<<<<<<3<=<<<;<<+
    seq1 274 T 23 ,.$....,,.,.,...,,,.,... 7<7;<;<<<<<<<<<=<;<;<<6
    seq1 275 A 23 ,$....,,.,.,...,,,.,...^l. <+;9*<<<<<<<<<=<<:;<<<<
    seq1 276 G 22 ...T,,.,.,...,,,.,.... 33;+<<7=7<<7<&<<1;<<6<
    seq1 277 T 22 ....,,.,.,.C.,,,.,..G. +7<;<<<<<<<&<=<<:;<<&<
    seq1 278 G 23 ....,,.,.,...,,,.,....^k. %38*<<;<7<<7<=<<<;<<<<<
    seq1 279 C 23 A..T,,.,.,...,,,.,..... ;75&<<<<<<<<<=<<<9<<:<<


    I don't understand the significance of the $ and ^ characters. The docs say they mark the start and end of read segments. Take the first line. Looking at the $, is that saying the that there is a read (the third read) whose last base is position 272 and the last base in that read is '.' which is the character after the $? Also looking at the ^ in row 1, is it also saying that the 24th read starts at position 272 and its first base is '.' which is the character after ^+

    It also says 'The ASCII of the character following `^' minus 33 gives the mapping quality.' What is the mapping quality? Is it the quality of the mapping of that read to the reference genome? So for row 1 the quality is 10 (43 for ascii code for + minus 33) and the mapping quality for row 4 is 16 (49 -33). I assumed this mapping quality information would be in the SAM file.

    If i am looking at variant data, is it safe to discard the $ and ^ data as I don't need to reconstruct the read sequence from pileup? I also don't think I see how I can use the mapping quality of a read segment to assess the quality of my variant data (please correct me if I am wrong!!!) - I can only meaningfully use the base qualities in the next column.

    Also, what is a reference skip which is mentioned in pileup user manual

    Having never used Samtools I am guessing there will be utilities to extract this data directly from the pileup file rather than my writing a custom parser?

    Thank you for your time

  • #2
    Have you tried using the mpileup using bcftools? it gives you a format that is nice and easy to read.

    Comment


    • #3
      How about the ">" or "<" characters in column 5 of the pileup format? I can't seem to find any information about what they signify:

      chr1 4483937 G G 48 0 60 7 .$....,. ?GEDA@D
      chr1 4483938 C C 45 0 60 6 ....,. H9GCFH
      chr1 4483939 T T 48 0 60 7 ....,.^~. JBH?FH@
      chr1 4483940 G G 48 0 60 7 ....,.. I<I:AHC
      chr1 4483941 C C 48 0 60 7 ....,.. I<IC?HC
      chr1 4483942 T T 48 0 60 7 ....,.. JCJC4HF
      chr1 4483943 C C 48 0 60 7 ....,.. J<JF>JF
      chr1 4483944 A A 21 0 60 8 ....,..^~C IAJHEJD?
      chr1 4483945 C C 36 0 60 8 >.>.<>.> JDJHEJE;
      chr1 4483946 T T 36 0 60 8 >.>.<>.> JFJHEJF;
      chr1 4483947 G G 39 0 60 9 >.>.<>.>^~. JAJIEJH;C
      chr1 4483948 T T 42 0 60 10 >.>.<>.>.^~, JEJAEJH;CE
      chr1 4483949 A A 42 0 60 10 >.>.<>.>., J9JEEJG;CJ
      chr1 4483950 A A 42 0 60 10 >.>.<>.>., JCJEEJH;FJ
      chr1 4483951 C C 42 0 60 10 >.>.<>.>., JFJ?EJG;FJ
      chr1 4483952 G G 39 0 60 10 >.>.<>.>., J)JCEJJ;FJ
      chr1 4483953 G G 42 0 60 10 >.>.<>.>., J?J@EJJ;FJ
      chr1 4483954 A A 42 0 60 10 >.>.<>.>., JDJFEJJ;FJ

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