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  • Calculating total no. of multimapped reads using samtools for RNA-Seq data analysis

    Dear all ,

    please excuse me if my questionaire is wrong.

    I am confused in estimating proper command for calculating number of multimapped reads using samtools for my paired end RNA-Seqdata, when i tried searching forums , i found two
    threads for calculating total no. of multimapped reads

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

    Application of sequencing to RNA analysis (RNA-Seq, whole transcriptome, SAGE, expression analysis, novel organism mining, splice variants)


    I tried using both , the results were very different , please suggest me proper command to calculate multimapped reads using samtools

  • #2
    I'd try filtering with FLAG 0x100 or 256. Sometimes I have seen inconsistencies depending on the mapper being used (e.g GEM mapper).

    Comment


    • #3
      I have used Tophat for mapping my genomes .

      Comment


      • #4
        If I recall correctly, I wasn't able to actually find this in the TopHat documentation, but take a look at this discussion. According to the top answer, the mapping quality encodes the number of mappings.

        255 = unique mapping
        3 = maps to 2 locations in the target
        1 = maps to 3-4 locations
        0 = maps to 5 or more locations (up to the number defined in "--prefilter-multihits")
        So I guess you could do something like

        Code:
        samtools view file.bam | awk '$5 < 255 {count++} END {print count}'
        Edit: this might be inefficient, I'm really not sure. But I think it'll get the job done.

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