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Old 05-30-2014, 01:03 PM   #5
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,238
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Quote:
We have run libraries with with insert sizes averaging as high as 1.1kb.
I wonder if it was amplicon library comprising same or similarly large sized fragments or was it a library with wide distribution of fragment sizes. I have sequenced such large sized fragment (amplicons) as well (in MiSeq) and I load 1.5x more than usual libraries to compensate for failed clusters. If all fragments are large, partially amplified clusters will be picked up by RTA and pass filter because RTA compensate for low signal intensity when most of clusters have low intensities. However, in library with wide size distribution, clusters from small fragments will have higher intensities and as a result low intensity clusters from large fragments will not pass. In widely distributed Nextera libraries with average 800 bp size I get fragments with 950 bp but they are small portion of sequences. All this indicates partial failure of large fragments’ clusters.

Quote:
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.
So, maybe it is not that small fragments are competing and displacing large ones (physically less feasible) but it is RTA operation that favours the high intensity small fragments clusters. I wonder if someone changes the recipes to allow for longer extension times during cluster generation if more large fragments will be sequenced.
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