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Sequencing a Low diversity library on the HiSeq

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  • Sequencing a Low diversity library on the HiSeq

    I am preparing a custom multiplexed library that will fall into the "low diversity" category. Low diversity meaning the first 5 nucleotides of read 1 will be identical among all clusters. There is a well known and well documented problem with cluster identification for low diversity libraries (outlined here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3030592/ ).

    The above paper and many of the comments on these forums refer specifically to the GAII, and suggest that spiking the library with 40-50% phiX control resolves the cluster calling issue.

    Now that the GAIIx has been all but phased out, I need to run my low diversity library on the HiSeq. The problem is that I don't know of anyone that has successfully run a low diversity library on a HiSeq, and my core informed me today that they have tried several times to run low diversity libraries but got awful results on the HiSeq, even after spiking with phiX %50.

    My question is, has anyone had success running low a diversity library on the HiSeq? If so, how did you manage to get it to work. Because my study does not require a massive number of reads, I am considering spiking my sample with up to 90-95% gDNA, hopefully drastically increasing the diversity and resolving cluster identification problems. Does anyone have experience running low diversity libraries on the HiSeq that could give me some advice?

    Thanks so much!

  • #2
    Is there a reason not to use a custom sequencing primer?

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    • #3
      I am sequencing viral DNA/gDNA integration junctions, so I am using that 5 nucleotides of viral DNA as a type of 'verification', that indeed a read contains the junction between viral DNA and gDNA, essentially showing that the sequencing primer is not mis-hybridizing to a similar (non-viral) sequence elsewhere in the genome. We are using a custom sequencing primer, but we prefer that it not hybridize up the the very edge of the viral DNA for reason described above.

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      • #4
        Can your core not run your samples by specifying a different lane (which is expected to have "normal" DNA) as the "control" lane for that run?

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        • #5
          Simcom: Judicious primer design coupled with the appropriate annealing temperature will virtually assure that the primer does not hybridize inappropriately.

          Genomax: the designation of a normal complexity sample as the control lane does not solve the problem (sadly). While it allows the signal thresholds to be set appropriately, it doesn't address the problem of cluster resolution in the low complexity lanes.

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          • #6
            Originally posted by GenoMax View Post
            Can your core not run your samples by specifying a different lane (which is expected to have "normal" DNA) as the "control" lane for that run?
            I think you are misunderstanding the problem. The issue is, because the first 5nt of read #1 are going to be all identical among clusters, and the machine uses these 5nt to call clusters, the machine has a hard time identifying/differentiating between different clusters (especially close overlapping clusters). So the reason for the gDNA is to add diversity to the sequence, allowing clusters to be called. Hence the reason it needs to be included in the same lane as the sample.

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            • #7
              There's an alternative approach, assuming that you have not yet constructed the libraries. Design them so the junction is at the opposite end of the insert, and perform paired-end sequencing. Cluster calling is based only on the first five cycles of read one, so you'll avoid the low-complexity issue.

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              • #8
                Originally posted by HESmith View Post
                Simcom: Judicious primer design coupled with the appropriate annealing temperature will virtually assure that the primer does not hybridize inappropriately.
                I agree with you that hybridization is unlikely, but if it does happen it will be indistinguishable from an actual integration. Among other things, we are interested in mapping low-abundance integrations, so if we aren't able to get the verification sequence on every read, we will likely need to go in and verify a subset of integrations manually, which may not be possible if an integration is present in only one cell for example.

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                • #9
                  Originally posted by HESmith View Post
                  There's an alternative approach, assuming that you have not yet constructed the libraries. Design them so the junction is at the opposite end of the insert, and perform paired-end sequencing. Cluster calling is based only on the first five cycles of read one, so you'll avoid the low-complexity issue.
                  Yep, exactly. Sadly my boss insisted on having a library that we can do single read OR paired end on (a money saving move potentially), so I had to design the junction on the first read side. And the samples are just about finished being prepped :/

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                  • #10
                    If, as you say, you can tolerate discarding 90-95% of the reads, then spiking in a gDNA library at that level will definitely solve your problem. After all, adapter dimers are often present at 5-10% in many libraries (the same % as your desired samples), and sequencing them is not a problem!

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                    • #11
                      Originally posted by HESmith View Post
                      If, as you say, you can tolerate discarding 90-95% of the reads, then spiking in a gDNA library at that level will definitely solve your problem. After all, adapter dimers are often present at 5-10% in many libraries (the same % as your desired samples), and sequencing them is not a problem!
                      Thanks, this gives me confidence. Just to be sure though: do the adapter-adapter ligation reads come back in the data, or does the machine throw them out and not include them in sequencing results? If you actually get the adapter-adapter reads from the machine, I should be golden.

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                      • #12
                        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.

                        --
                        Phillip

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                        • #13
                          Yes, adapter reads are present.

                          Just remember that you'll also need to include the standard sequencing primer for the gDNA library (or construct the gDNA library with custom adapters to match your custom primer).

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                          • #14
                            Originally posted by HESmith View Post
                            Yes, adapter reads are present.

                            Just remember that you'll also need to include the standard sequencing primer for the gDNA library (or construct the gDNA library with custom adapters to match your custom primer).
                            Yep, I planned on including both primers. Thank you so much for your help, I really appreciate it.

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                            • #15
                              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.

                              --
                              Phillip
                              OK, that is good to hear. I'm not sure why my core was having trouble spiking 1:1 gDNA.

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