[nucacidhunter]

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.

[austinso]

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

[DNATECH]

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.

1.5% index hopping with no adapters spiked in. Does that mean anything?

[nucacidhunter]

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.

[kmcarr]

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.