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  • #16
    Originally posted by gringer View Post
    1.5% index hopping with no adapters spiked in. Does that mean anything?
    I think 1.5% hopping is from the libraries in the pool and not the spiked-in adapters.

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    • #17
      Makes me wonder if single molecule consensus is out of the question, since it would seem that the mitigation strategy requires defined barcode sequences.

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      • #18
        It shows that PCR-free libraries can hide a larger amount of adapters and adapter dimers somehow entangled in the y-adapters. We have several times seen surprises with (on the BA) clean looking PCR-free libraries in this regard.

        Originally posted by gringer View Post
        1.5% index hopping with no adapters spiked in. Does that mean anything?

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        • #19
          Originally posted by ECO View Post
          Anyone doing significant multiplexing should be using dual indicies, which reduces this problem dramatically (making swapped reads go to Undetermined while demultiplexing).

          Interested to see if this is a problem in our novaseq runs...will update if I note something.
          I wonder if there is any update on this.

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          • #20
            Originally posted by nucacidhunter View Post
            I wonder if there is any update on this.
            In our core we did the following test: Project with 64 RNA-Seq libraries prepared using Illumina TruSeq Stranded mRNA HT Dual Index kit on a Perkin Elmer Sciclone G3 robot. We also add a second AMPureXP cleanup at the end of the prep. We divided these into 8 pools of 8 libraries each following the dual index pooling guidelines in Illumina's white paper about index hoping so that each pool had 8 dual-unique index pairs. Each pool was loaded on one lane of HiSeq 4K flow cell and sequenced SE50 (51 cycles R1, 8 cycles I1, 8 cycles I2) using standard protocol.

            Total output per lane was ~370-380M reads. With 8 i7 and 8 i5 indexes in each lane there are a total of 64 combinations possible, 8 expected combinations and 56 which may result from index hoping. I ran bcl2fastq (2.19.0) looking for all index pairs, permitting 1 mismatch per barcode.

            For any given unexpected index pair produced through index hoping I found an average of ~35,000 (± 9,850) reads or approximately 0.01% of the total lane output.

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