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  • #31
    Hi all,

    I am not sure if I should ask it here, still new to NGS, and I have lots of questions that I couldn't figured out.

    1) I am using a custom adapter (that contains p5 p7 for flow cell binding), custom sequencing primer and also a custom index i7 primer for my work. I come across this dual-indexing, I am just wondering if it is possible to have a custom index i5 primer?

    2) I am not familiar with Nextera, why is the dark cycles necessary? why not just custom make an index i5 primer that spam the 7bp constant read and read directly into the index? or wait did I miss out something. where is this index i5 primer primed from?

    3) must it be index read 1 = i7 index; index read 2 = i5 index? can't be other way round? it's still on the same strand right?

    sorry if I asked too many of the dumb questions.

    cheers.

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    • #32
      1) You should be able to design an i5 index primer/adaptor
      2) The i5 index is primed and read from the P5' oligo that's grafted onto the flowcell for clustering. No custom read primer is needed. However, due to the way linearisation is performed during cluster-gen. 7 dark cycles are needed to extend this oligo until the i5 index is reached. See page 7 of this document (http://supportres.illumina.com/docum...15032071_b.pdf)
      3) Read sequence goes as follows: Read 1, i7 index, i5 index, Read 2. Everything but read 2 is read off of one strand (to allow for dual indexing on single read runs without the need to resynthesise the second strand).

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      • #33
        High multiplexing of Nextera libraries

        I would like to reopen this thread, as we have successfully run a pool of over 1400 samples (plasmids) on a MiSeq using our own i5 and i7 index primers. We reduced the volume of tagmentation considerably and developed a PCR protocol to attach unique combinations of our own i5 and i7 primers to each sample. The MiSeq had no trouble demultiplexing these 1400 samples. We hope to publish the methods soon. However, we have since tried running over 4000 samples in a pool and here the MiSeq had big trouble demultiplexing. Has anyone out there tried such high levels of multiplexing? If so, I would like to hear about your experience.

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        • #34
          Originally posted by creeves View Post
          However, we have since tried running over 4000 samples in a pool and here the MiSeq had big trouble demultiplexing. Has anyone out there tried such high levels of multiplexing? If so, I would like to hear about your experience.
          You should be doing the demultiplexing off-line using bcl2fastq/CASAVA when you are doing something so outside "normal"

          What was the length of the tag reads BTW.

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          • #35
            Well done creeves on pushing the multiplexing so far. What was the edit distance between your barcodes? The default parameters for demultiplexing tolerate 1 mismatch in each barcode I believe. If some of the barcodes have an edit distance less than 2 you might need to demultiplex looking for exact barcode matches.

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            • #36
              Bar codes

              The 8-base i5 and i7 indices had a Hamming distance of at least three from all others.

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              • #37
                Was this V2/V3 chemistry and what was the cluster concentration? I wonder if the problems you are having are because of overloading and not number of samples.

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                • #38
                  We have done up to 1536 multiplexing in a run so far. Demultiplexing by the MiSeq software worked well.
                  However, there is a bug in the current RTA version: There is a timeout for writing fastqs in the output folder. For large V3 runs with many samples, part of the fastqs do not end up in the output folder. They are however in the analysis folder.

                  Meanwhile we published our dual indexing strategy - in case anyone is interested:
                  BMC Genomics - Cost-efficient high-throughput HLA typing by MiSeq amplicon sequencing

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                  • #39
                    I have studied this thread of posts, but I still have a few questions that confuse me.

                    I understand that the p5 connected fragments are grafted at the uracil site after bridging amplification but before the first read. My question is that, after the turnaround, there would be both p5 and p7 connected fragments. Does the p7 connected fragments need to be cut off or removed before the second read? If so, how does it carried out?

                    Comment


                    • #40
                      If I understand your question, it suggests you do not understand the process of cluster formation. After denaturing the library fragments for loading, one strand can hybridize to the p5 oligo on the flow cell and the other can hybridize to the p7 oligo. Bridge amplification can proceed for both strands as long as the strands come from a library fragment with p5 on one end and p7 on the other. If a strand can hybridize to p5, the p7 end of that strand is not complementary to the p7 oligo on the flow cell. Likewise, if a strand can hybridize to p7, the p5 end is not complementary to the p5 oligo on the flow cell. However, once the first extension is complete for either case and the untethered strand is washed away, the distal end is now able to hybridize to the other oligo on the flow cell. Whichever end first hybridizes (p5 or p7) the cluster ends up being an equal mixture of complementary strands tethered at the p5 or p7 ends.

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                      • #41
                        Illumina has a simplified animation for cluster generation/sequencing available here: http://www.youtube.com/watch?v=HMyCqWhwB8E

                        Comment


                        • #42
                          Hi, Creeves, thank you for your reply. I understand that after the cluster generation, there would be a mixture of complementary strands grow from the p5 or p7 ends. Some documents suggested the strands with p5 ends attached to the flow cell would be stripped off for the first read and the i7 index read to ensures "that all copies are sequenced in the same direction". Then, after the turn around, the p7 grafted strands would be cleaved to allow all reads of read 2 come off the p5 tethered stands. Does this sound correct? If it is correct, I'd like to know how the p5 and p7 tethered stands were cut?

                          Here is the link where I got the information:

                          Comment


                          • #43
                            Thank you, GenoMax. I am ashamed to say that I can't open any youtube link, because I am in China now. If anyone could explain to me how it works in simple words, it'll be greatly appreciated. I am sorry for being so ignorant.

                            Comment


                            • #44
                              Indeed, my customized adapter and sequencing primer is the same for read1 and read2. So if the sequencing is done from one direction, it would be fine. But if the cluster is composed of two opposite strands when sequencing is done, then my sequencing primer could anneal to both stands and my reads would be a mess. I'd really like to find out if the opposite stands are clipped off from the cluster for both read 1 and read2? Thanks

                              Comment


                              • #45
                                Since you can't see the video how about this link: http://nextgen.mgh.harvard.edu/IlluminaChemistry.html

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