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  • Mapping statistics from fragmented FFPE samples using Ribo-Zero

    Recently we have performed RNA sequencing using Ribo-Zero in order to remove rRNA from fragmented FFPE samples. The number of reads passing filter are around 30 million per sample. The samples were mapped using Tophat and the readcounts were calculated using HTSeq-count.There aren't much reads left that map to the ribosomal RNA, which is a good thing, but the number of reads that were assigned to a gene according HTSeq-count were not as high as we hoped to see (3-12 million reads per sample).

    It seems that there are many reads that do not map to unique locations and a lot of unmapped reads do map to human (BAC) DNA clones which results in a loss of >60% of the reads that were passing filter.
    However, the reads that are assigned to do show a very nice profile (compared to DSN-treatment, which we used to use).

    Does anyone experience the same?

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