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  • Changing dNTP Flow Order for Low-Complexity Template Regions

    I've had this recurring idea that I've wanted to try, but wanted to seek thoughts from everyone here first.

    Let's say I'm sequencing a template that I know has a low complexity region near the 5' end. For example, say it's an amplicon library with a constant 20mer sequence (call it S1) directly after the P1 Ion Library sequence, that corresponds to the priming site on my amplicons. What I'm curious to know is, if I set the dNTP flow sequence during sequencing to be the same as my sequence over the first 20 flows, will the PGM simply read through my S1 sequence faster, or will it cough and think something is wrong because it is getting signal every flow over that first 20?

    This idea could be generalized a little further, if say you know the sequence is skewed in your library, you could tweak the entire dNTP flow sequence for the whole run, so that you'd be getting relative more bases in fewer flows.

    Does this make sense, or are there some other considerations I'm not taking account of?

  • #2
    Assume you can change the flow order. The current filtering algorithm by default take 60% incorporation rate as cutoff. Based on your model, incorporation rate is almost 100%, which will be automatically filtered out unless you change the filter to 100% but you might end up with many polycolonal beads.

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    • #3
      Sure, that the high rate of dNTP incorporation would make the machine think that something was wrong. So maybe the better question is whether the 60% incorporation threshold is a parameter that can be changed?

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      • #4
        Originally posted by SeqNerd View Post
        Sure, that the high rate of dNTP incorporation would make the machine think that something was wrong. So maybe the better question is whether the 60% incorporation threshold is a parameter that can be changed?
        Even you can change the filter, how can you filter out polycolonal beads then?

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        • #5
          Good point. I guess I assumed (in the case of sequencing through a constant region) that reads not matching the intended pattern corresponding to the specified dNTP sequence would have to happen at the mapping step.

          Do you know if the incorporation % is the only metric used to identify polyclonal beads? I don't know how close to saturated the beads are, or how the pH response curve depends on the saturation on the bead, but maybe there is a decrease in signal per nucleotide if half of the DNA on the bead has one sequence and half another.

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          • #6
            IonTorrent is in fact experimenting with alternate flow orders, see http://flxlexblog.wordpress.com/2011...ets-the-stage/

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            • #7
              On a related note, how does the PGM handle every library element having the same run of sequence? We are having balancing issues due to low complexity on the HiSeq and lose quite a bit of data unless we put in other samples or a high percentage of phiX.

              If the PGM can sequence well one amplicon, that would be very useful. I can see where flowing the dNTP order to match the priming site would speed up the sequencing to the internal space, maybe even increase read length?

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              • #8
                We're sequencing constructs that start with a 6mer barcode followed by a standard sequence and haven't seen any problems with the lack of sequence diversity in the reads.

                I think the Ion Torrent basecaller calibrates itself differently than Illumina's- each read starts with a key (library or control sequence) that calibrates the basecaller for that individual well. I think that eliminates much of the need for a chip-wide calibration requiring a diversity of basecalls.

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                • #9
                  Illumina's problem with low complexity I believe is that it is used in the cluster identification stage; it wants a certain degree of variability so it knows to divide two regions of signal into distinct clusters.

                  Ion Torrent has no cluster calling issue, since the sequences come from a defined grid of sensors. Hence, no issue.

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                  • #10
                    Originally posted by flxlex View Post
                    IonTorrent is in fact experimenting with alternate flow orders, see http://flxlexblog.wordpress.com/2011...ets-the-stage/
                    Nice post - but had you seen the code name for the new Ion Torrent flow order?

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