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  • Low Exome Coverage with Truseq DNA Exome and NextSeq 500 workflow?

    Dear All,

    I studied a whole exome with Truseq DNA Exome (2x75 and suitable High output kit) on NextSeq 500.. AFter sequencing I got around 46 million reads per sample and 42 million of these were mapped as globally.

    As you guess I did BamQC report and got that only 11 Milllion of these 46 million reads are mapped to the targets of TruSeq DNA Exome kit. And I have used the bed file provided by Illumina for this kit.

    So what could be the possible problem that On global Nearly 90% of my reads are mapped but in target only around 23% of the reads are mapped?

    Thanks in advance

  • #2
    It indicates that on target reads (enrichment) is low and most of reads are from background. This figure for most kits is above 70%. At least one step in target capture has been non-optimal.

    I wonder what is the duplication rate.
    Last edited by nucacidhunter; 12-17-2018, 11:43 PM.

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    • #3
      Originally posted by nucacidhunter View Post
      It indicates that on target reads (enrichment) is low and most of reads are from background. This figure for most kits is above 70%. At least one step in target capture has been non-optimal.
      Thanks for the answer. So could you suggest anything with that? Because I have been following the exact steps of the kits checklist guide.

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      • #4
        Possible important issues:

        1- degraded or low concentration of blocking oligos
        2- inefficient hybridisation step (temperature, time, reaction composition)
        2- non-stringent wash steps

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