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Old 05-29-2014, 04:37 PM   #3
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,238
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For reasons unclear to me, short amplicons seem to cluster vastly better than longer amplicons.
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.

Last edited by nucacidhunter; 05-29-2014 at 05:18 PM.
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