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  • Ion Torrent targeted sequencing

    Hello Every one,

    I am a new NGS user. I am starting to work on targeted sequencing using Ion Torrent technology.

    My targeted gene is BRCA1 and I have about 50 samples. The gene is 112 amplicon. I would like to know how many samples per run I can pool?

    Thank you for your help

  • #2
    That depends a little on your experiment, ie how big your amplicons are, if its germline somatic mutationd and which chip you want to use ie 314/316/318.
    FYI -- we have recently been testing Sabioscience (Qiagen) gene-read (multiplex PCR) for this type of targeted experiment, seems to work well. We estimate (guess-timate) that 48 samples should be reasonable on a 316 (200mbp) Ion run.

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    • #3
      Thank you for your replay.
      I was just wondering how do you do these calculations?

      I found out that if I used 318 I will get a coverage of 7,84 but should use tow chips as the chip takes 48 samples. on the other hand if I used 316 I will have a coverage of 1,57. My samples are DNA from blood, do I need still (big) coverage?

      Comment


      • #4
        Here's a back-of-the-envelope calculation (i.e. it's rough).

        Assuming that average amplicon size = 200 bp
        you have 112 amplicons,
        you want 100X seq coverage

        = 2,2 mbp/individual (ie 200bp X 112 amplicons X 100 fold).

        2,2 X 48 = 107 mbp total. ABout capacity of a 316 mbp

        Very rough though so maybe overestimate needed coverage in order to achieve the minimum you need (ie difference in amplicons, barcodes etc).

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        • #5
          Thank you

          Comment

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