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Old 06-02-2014, 04:37 AM   #6
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Originally Posted by nucacidhunter View Post
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.
No, I meant "amplicon" in the general sense. This was a genomic DNA library.
Not sure what is happening with your actual amplicon libraries. But in our case, we just used the concentration based on qPCR and nailed the cluster density.

Originally Posted by nucacidhunter View Post
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.
Nope, as I write above, this does not fit with the actual results we got.

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