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  • Samtools flagstat - no duplicates?

    I'm having some problems with samtools flagstat (which surprisingly should be straightforward as it has no parameters that can be wrongly defined). Samtools flagstat is used to return summaries of the flags produced within bam files. I was given intensities from exome sequencing, converted them into fastq files and removed reads with low quality (fastq format). I ran an alignment on fastq files using BWA, and got the following weird results from flagstat:

    109638024 + 0 in total (QC-passed reads + QC-failed reads)
    0 + 0 duplicates
    108394681 + 0 mapped (98.87%:nan%)
    109638024 + 0 paired in sequencing
    54819012 + 0 read1
    54819012 + 0 read2
    106794832 + 0 properly paired (97.41%:nan%)
    108037629 + 0 with itself and mate mapped
    357052 + 0 singletons (0.33%:nan%)
    1071345 + 0 with mate mapped to a different chr
    977974 + 0 with mate mapped to a different chr (mapQ>=5)

    Common biological knowledge dictates that every sequencing should produce 10-15% PCR or optical duplicates, which is why I was surprised when I got 0 duplicates (I tried it on several samples in the same run, and they all gave 0 + 0 duplicates, which makes me feel something is wrong with flagstat). It got even weirder after I did samtools rmdup and retrieved the flagstat for that bam file:

    59158619 + 0 in total (QC-passed reads + QC-failed reads)
    0 + 0 duplicates
    57915276 + 0 mapped (97.90%:nan%)
    59158619 + 0 paired in sequencing
    26999098 + 0 read1
    32159521 + 0 read2
    56923778 + 0 properly paired (96.22%:nan%)
    57745533 + 0 with itself and mate mapped
    169743 + 0 singletons (0.29%:nan%)
    727165 + 0 with mate mapped to a different chr
    672895 + 0 with mate mapped to a different chr (mapQ>=5)

    So about half of the original reads are kept, i.e. there are some duplicates, but they were not flagged in bwa. How could this have happened?
    "Though it may seem that all's been said and done, originality still lives on" - some unoriginal guy who had nothing better to write as his signature

  • #2
    You'll only get duplicates in the BAM file if they are marked as duplicates. BWA doesn't mark them and samtools rmdup removes them. Try using picard MarkDuplicates

    java -jar ${picard_dir}/MarkDuplicates.jar I=${name}_bwa.bam O=${name}_bwa_dedup.bam M=${name}_bwa_dedup_metrics.txt

    Chris

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    • #3
      Well, I have considered a dodgy trick: if I subtract the number of reads in the bam rmdup file from the number of reads in the original bam file, I should get back the number of duplicates removed, right?

      One thing I'm still not certain about: BWA aligns the reads to get a bam file, and it doesn't flag any duplicates. Does rmdup than find those duplicates without them being previously marked/flagged? If so, how is this done?
      "Though it may seem that all's been said and done, originality still lives on" - some unoriginal guy who had nothing better to write as his signature

      Comment


      • #4
        It looks for reads that have the same start position and then keeps the best one based on sequence quality scores - it's a fairly basic approach. Also look at this thread which recommends to use picard:

        Discussion of next-gen sequencing related bioinformatics: resources, algorithms, open source efforts, etc


        BWA is kept light weight and doesn't do things like marking duplicates or even making BAM files as some applications don't need to mark duplicates - e.g., in ChIP-seq.

        Chris

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