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  • Tophat paired end read

    Hi,
    I am analyzing RNA-Seq data from one published paper.

    Illumina Hiseq 2000 paired end reads. each end read is 101 bp.

    I picked up one pair of reads to check the inner distance (required for Tophat) between these two reads, and found out that these two reads are actually much overlapped.

    Then I realized that the size of the selected frangments might be around 133bp, which is why these two paired end reads have overlapping.

    In this case, what should we type in for the inner distance between reads when using Tophat? "0" ?

    hope you understand my question. Looking forward to hearing from you.

  • #2
    The mean inner distance will be your mean insert size - the length of your two reads. Or if you instead have the length of your size selected fragments it will be fragment size - 2 * read length - adaptor lengths.

    I suggest you map your paired end reads to the transcriptome with a non splice aligner, like bwa or bowtie. Then calculate the mean insert size with the help of picard tools. Subtract the reads and you have your inner distance. If the reads are overlapping then your inner distance will be negative.

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