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  • #31
    Update--
    Quality on first read was low and got an "N" in up to half the reads at positions 12, 20, 21, and 24. Indexing read was fine. The instrument completely lost focus on 2nd read (100 nt read) so I got nothing. That read started reading into the specific amplicon reverse primer which I thought would be okay because clusters had been found on the first read. Apparently not true. Doesn't matter for this library but eventually want to join longer reads. Am trying again next run with 20% spike in of high complexity library and 50% lower cluster density.

    We've had the solenoid change-out and the s/w upgrades (just before this run) and bubble-related loss of focus should not be entire lane starting with cycle 1 (or so Illumina claims). This was 100% loss, not a few percent.
    Attached Files
    Last edited by HMorrison; 12-02-2011, 09:23 AM.

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    • #32
      Originally posted by HMorrison View Post
      Update--
      Quality on first read was low and got an "N" in up to half the reads at positions 12, 20, 21, and 24. Indexing read was fine. The instrument completely lost focus on 2nd read (100 nt read) so I got nothing. That read started reading into the specific amplicon reverse primer which I thought would be okay because clusters had been found on the first read. Apparently not true. Doesn't matter for this library but eventually want to join longer reads. Am trying again next run with 20% spike in of high complexity library and 50% lower cluster density.
      Actually you likely got burned by one of the recent updates. Until the v3 chemistry update, the instrument did not refocus after strand turnaround. But now it does. If the instrument had used the focal points it was using during the successful index read, you might have been okay.

      Thanks for the info, though.

      If this happens again, Tech Support may be able to guide you through a forced refocus of the instrument after the cycle where your adapter ends. (I think it mainly would involve halting the run and re-starting it.)

      Hmm, if that works, it could be a general (although drastic) solution to low complexity bases even in the first read. Just let as many cycles as necessary complete (ignoring the possibly terrible results you are getting). Stop the run. Start a completely new run on the same flow cell, from that point. At 4 bases from the end your low complexity start, cluster calling would happen.

      --
      Phillip

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      • #33
        Hello,

        I am glad to see a thread with so many helpful informations for us dealing with low complexity samples! I am having the same problems - 5-11 nt label at the beginning of the read. So far the quality has been very disappointing and it worsened dramatically when we switched to HiSeQ in spite of 50% phiX spike-in and lower cluster densities. We were also not very happy with our sequencing facility for other reasons. Those of you who had good experience with sequencing low diversity samples - can you please share which sequencing facilities seem to do a good job of it? In our case some preparation is very time consuming and costly and we do not want to loose any more data...

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        • #34
          Mixed success

          Still haven't gotten this to work well--instrument is being checked out right now by Illumina engineers. They say there always was refocusing on read two (really 3; after indexing read) so I needed randomness at first few bases there, too. PhiX spike in didn't help much. Think I need to redesign primers to stagger the signal.

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          • #35
            Originally posted by HMorrison View Post
            Still haven't gotten this to work well--instrument is being checked out right now by Illumina engineers. They say there always was refocusing on read two (really 3; after indexing read) so I needed randomness at first few bases there, too. PhiX spike in didn't help much. Think I need to redesign primers to stagger the signal.
            Hillary,

            we have had no success so far, either :-( So your 20% spike-in phiX run with 50% normal cluster density was not a success, either? What specific cluster density did you used? Was it chemistry v3? We will be doing another attempt on HiSeQ with v3 chemistry in a couple of weeks so I am very interested in the technical details. TIA

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            • #36
              [QUOTE=pmiguel;57185]We got it to work on our HiScanSQ -- which uses the same chemistry as the HiSeq, but only scans the top of the flowcell. Not an identical situation, but we had some SMART cDNAs that we sheared and ligated TruSeq adapters on. So about 1/2 of them had the same 50 nt of SMART primer at the beginning. We mixed them 1:1 with a genomic DNA library. Cluster registration went fine.

              --


              We are also considering this, may I ask you why half of the reads had the smart oligo at the beginning (since you radomly sheared them)

              Thanks and best

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              • #37
                we sequenced low complexity/diversity libraries by mixing them in with other experiments of high complexity. this way low complexity clustered can be indentifed in amongst high complexity clusters

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                • #38
                  If you only have a strong initial bias but with diverse sequence afterwards there is a somewhat dramatic solution which does work on the Hi-Seq, but unfortunately has to be applied to the whole run.

                  Basically you start read 1 as normal, but before starting to do imaging you do 5 chemistry cycles without doing any imaging (so called 'dark cycles'). You then do the run as normal and read through the rest of your sequence. At the end of the first read you strip the templates and then re-anneal primer 1 and do a 5 cycle read 2 to get the biased bases at the start. You therefore end up with all of the sequence you want, but spread out over 2 reads instead of 1.

                  Having said that, in your case that's not what I'd do. Given that you're only using the first 5bp to confirm that you have the correct sequence I'd be tempted to use a custom primer to prime over the biased positions though. If you have a different sequence at the start the primer shouldn't anneal properly so you should only get signal from clusters with the correct sequence.

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                  • #39
                    Originally posted by josdegraaf View Post
                    Originally posted by pmiguel View Post
                    We got it to work on our HiScanSQ -- which uses the same chemistry as the HiSeq, but only scans the top of the flowcell. Not an identical situation, but we had some SMART cDNAs that we sheared and ligated TruSeq adapters on. So about 1/2 of them had the same 50 nt of SMART primer at the beginning. We mixed them 1:1 with a genomic DNA library. Cluster registration went fine.
                    We are also considering this, may I ask you why half of the reads had the smart oligo at the beginning (since you radomly sheared them)

                    Thanks and best
                    Will have to recall from long term memory.

                    Okay, the cDNAs all were flanked by these 50 bp SMART adapters. These cDNAs were also fairly short prior to shearing for reasons I won't go into. They were fragmented to roughly 1/2 their initial length. Then "end polished" and ligated to Illumina adapters, probably using the TruSeq DNA prep kit. Hence, random chance put 50% of the reads adjacent to the SMART adapter.

                    SMART is designed to amplify full-length cDNAs using the ingenious "PCR Suppression" method. So if your post-shearing length is a small enough fraction of the amplified cDNA length, it should not be much of an issue. Possible to further reduce contribution towards your sequence from adapter using a rare-cutting restriction enzyme site in your SMART adapter. Not sure it would be worth it, though.

                    --
                    Phillip

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