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  • how to calculate RPKM from SAM

    i am very new to bioinformatics and been trying to learn new things every day.

    this time, i need to find out RPKM from a sam file. i mapped RNAseq reads to refMrna and have a sam file generated. the data was single end and had length 36. from the sam file, i took the number of unique counts of all genes that were mapped. now i have a count file with gene name and its count.
    eg.
    NM_000172 31239
    NM_000242 26765
    NM_000265 5160
    NM_000267 28499
    NM_000356 27352

    the RPKM formula suggests..
    (number of reads mapped to a gene/ (totat number of reads X read length)) X 10^9

    so, for the first read mapped, NM_000172, the number of reads mapped is 31239, read length is 36.
    but what will be the total number of reads ?? is it all the reads that were mapped in sam file. including flags like *
    please guide

  • #2
    If you used 'TopHat' for mapping, use 'cufflinks' to quantify expression in RPKM.

    Comment


    • #3
      Your formula for RPKM is wrong. Replace read length with transcript length. So you'll need to calculate:

      (number of reads mapped to a transcript) / (totat number of reads X transcript length) X 10^9

      It looks like you've already got the read count. The total number of reads is the number of reads mapped in your sam file. You can check this by using samtools flagstat. If you can obtain the number of bases for each of those genes, you'll be all set.

      I can't anything meaningful about the "*" flag.

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