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  • Problem Picard "Alignment start should != 0 because reference name != *"

    Hello,

    Using Picard Validation, I obtain this message for some reads : Alignment start should != 0 because reference name != *
    If I grep the read in the SAM file, I can see that the read is mapped (there is the name of a reference scaffold in the third column) but the position (column 4) is 0.
    This is very strange, how can this happen ?
    Do you know how to fix this ?

    Thanks,

    Muriel

  • #2
    What's the flag on that read?

    Comment


    • #3
      Hello,

      After doing bwa aln and bwa sampe, I obtain a sam file and I use Picard ValidateSamFile.
      The errors are :
      ERROR: Record 38208, Read name HWI-ST1206:14:C296WACXX:6:1101:8672:9595, Mate negative strand flag does not match read negative strand flag of mate
      ERROR: Record 40751, Read name HWI-ST1206:14:C296WACXX:6:1101:8820:10033, Alignment start should != 0 because reference name != *.
      If I grep the read with the second problem, here is what I obtain :
      HWI-ST1206:14:C296WACXX:6:1101:8820:10033 113 gi|300388125|ref|NC_013991.2| 0 25 46M =176 215 GTACTTTTTTTTTTAGTATTTTAATTTTGTATTTATTATTTATATT >GCGAGGEIIIHHGHHIHE@HHC:CA<<AAGC>DFHFFDDDDD@@<RG:Z:Pa1370-Seq1 XT:A:U NM:i:0 SM:i:25 AM:i:25 X0:i:1 X1:i:1 XM:i:3 XO:i:0 XG:i:0 MD:Z:0 XA:Z:gi|300388125|ref|NC_013991.2|,-86195,4M1D42M,1;

      HWI-ST1206:14:C296WACXX:6:1101:8820:10033 177 gi|300388125|ref|NC_013991.2| 176 37 85M =0 -215 GCAGCTAGGTCTAGAGGGAAGTTGTGAGCATTACGTTCATGCATTACTTCCATACCAAGGTTAGCACGGTTGATGATACCAGCCC @@@@@>@@>;;;AA=>BBBA<BBBBAA:BA=;B=9??ABBBBBBBBAA>BA<?*BABBB>C?AC7<CC@>A>72BC@@)=<A=== RG:Z:Pa1370-Seq1 XT:A:UNM:i:1 SM:i:37 AM:i:25 X0:i:1 X1:i:0 XM:i:1 XO:i:0 XG:i:0 MD:Z:78T6
      Any idea on how I can have a read map but no position given ???

      Thanks !

      Muriel

      Comment


      • #4
        That would seem to be a bug.

        Comment


        • #5
          OK thanks !
          Any idea on what I can do ?

          Comment


          • #6
            Try emailing the list.

            Comment


            • #7
              OK, i will, thanks for your help !
              Muriel

              Comment


              • #8
                Hi Muriel,

                I am facing the same problem. Did you find a solution for it?

                Thanks and kind regards

                Comment


                • #9
                  Hello !
                  Well, what I did is a personal arrangement. I should probably write to the mailing list, I don't remember if I did or not ahah !!
                  The problem is that some read are supposedly mapped on the reference because there is a scaffold name in the column 3, but the starting position (column 4) of the read is 0 and this is the problem.
                  Here is what I did :

                  ### List reads that have "Alignment start should != 0 because reference name != *"
                  samtools view -h input.bam | awk 'BEGIN {OFS="\t";} {if ($1=="^@") {print $0;} else {if ($4==0 && $3!="*") {$4=1; print $1>"read_file"} if ($8==0 && $7!="*") {$8=1;print $1>"read_file"} print $0;}}' | samtools view -bS - > input_OK.bam ; sort read_file | uniq > read_file_sorted

                  ### Exclude problematic reads
                  #java -Xmx20g -jar FilterSamReads.jar INPUT=INPUT_OK.bam FILTER=excludeReadList READ_LIST_FILE=read_file_sorted OUTPUT=output.bam

                  Good luck !

                  Muriel

                  Comment


                  • #10
                    Hi Muriel,

                    THanks for your reply!!

                    Kind regards

                    Comment

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