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Old 08-01-2017, 01:35 PM   #111
pmiguel
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Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
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Quote:
Originally Posted by nucacidhunter View Post
Index hopping is the result of annealed oligo extension by ExAmp. I do not know the details of ExAmp but KAPA HiFi polymerase under stringent cycling condition is able to extend primers as long as the 3 base and other 6 bases in the 10 base region of 3 is complementary even though the rest of the oligo is not a match and just hangs off the template.

Left over adapter oligos, PCR primers, single-adapted and non-adapted fragments can act as oligo and result in index hopping, neutral and cluster forming fusion fragments, respectively. So presence of high concentration of oligos acting as primers and longer incubation of library pool will increase these artifacts. I also would expect to see more fusion with PCR-free libraries as the proportion of fragments without adapters in both end are higher in comparison to PCR amplified libraries.
I hope not! That would also tend to create massive amounts of chimerism due to repetitive elements in genomic DNA, for instance. Hopefully whomever designed ExAmp would not allow low-stringency interactions of the sort you describe for the KAPA "HiFi" polymerase to result in this sort of (undesired) recombination.

I'm not really following why we need to posit either low stringency annealing event nor actual ligations (as the mechanism described in Brian's post would require) to explain index hopping. If there is any amplification occurring anywhere but tethered to the surface of the flowcell, then unincorporated adapter oligos could anneal and be extended, creating a "cross-over event" that would generate an index hopped library molecule. If that molecule seeded a cluster, then we would have an index hop.

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Phillip
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