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Old 05-30-2014, 08:00 AM   #4
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Originally Posted by nucacidhunter View Post
I think preferential clustering of smaller fragments (amplicons) can be explained by bridge amplification process. Library templates are denatured and mixed with hybridisation buffer. At this stage we expect that denatured fragments will stay single stranded and stretched free of secondary structure. If there is hybridization of strands back to their complementary strands at this step, it thermodynamically will be in favour of smaller fragments, hence reducing their number for next step not the long fragments. At next step, denatured fragments are pumped through flow cell lane and every fragments will have relatively equal chance of hybridization to oligo lawn on flow cell surface because they (should) have the complementary adapter sequences. After hybridisation, bridge is formed and amplification mix is pumped through to synthesize complementary strand to bridged fragments. Extension time is quite limited (15 sec in MiSeq) and at this step large fragments are less likely to have end to end synthesis of complementary strands because of pause or dropping out of polymerase. The result would be that those fragments will not be amplified in the next round of amplification (I think there is 30 cycle or so ) and will form weak clusters (if any) with low strand numbers depending on in which cycle this happens. Contrary, small fragments will have high chance of complementary strand synthesis end-to-end and therefore will dominate the properly formed clusters which will produce strong signals for RTA to detect and pass them.
Yeah, sound reasonable, but does not gibe with results I have seen. We have run libraries with with insert sizes averaging as high as 1.1kb. (Estimated by mapping pairs back to a reference after sequencing.) qPCR accurately predicted the expected cluster density. Which it would not have if there had been a high level of failure by the polymerase to complete product strand synthesis.

So, whatever the explanation, it needs to account for this. Which leads one to think there must be some sort of competition among amplicons. Something that would allow the shorter amplicons to displace the longer ones and prevent them from creating clusters. That way, if the shorter amplicons are removed, the longer amplicons can form good clusters. However I can't think of a reasonable mechanism of competition. So maybe something else is going on?

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