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  • Opusone
    Junior Member
    • Apr 2013
    • 1

    #1

    newbie BWA alignment question

    another newbie question - I am trying to use BWA to align paired end reads to human ref genome.

    here is the sequence of what i did -
    1) alignment
    bwa align ref fq1 > 1.sai

    2) make sam
    bwi sampe ref 1.sai 2.sai fq1 fq2 > .sam

    3) convert to bam
    samtools view -S -b .sam -o .bam

    4) sort bam
    samtools sort .bam .sorted

    5) index bam
    samtools index .sorted

    6) check stats
    samtools flagstat .bam > .stat

    everything finished running without any error, but when i check the .stat file, this is what i see

    0 + 0 duplicates
    0 + 0 mapped (0.00%:nan%)
    61091524 + 0 paired in sequencing
    30545762 + 0 read1
    30545762 + 0 read2
    0 + 0 properly paired (0.00%:nan%)
    0 + 0 with itself and mate mapped
    0 + 0 singletons (0.00%:nan%)
    0 + 0 with mate mapped to a different chr
    0 + 0 with mate mapped to a different chr (mapQ>=5)

    in every sample, there is very few to no aligned reads. can anyone comment on what could possibly be the issue here and if there is a way to check the output after each stage in this process. thanks.
  • swbarnes2
    Senior Member
    • May 2008
    • 910

    #2
    .sams are human readable. So start by looking at those.

    You did index the genome too, right?

    Comment

    • MBekritsky
      Member
      • Nov 2009
      • 15

      #3
      Did you align the second set of fastq sequences? All I see is
      bwa align ref fq1 > 1.sai
      but not
      bwa align ref fq2 > 2.sai

      Comment

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