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Old 02-22-2015, 01:39 PM   #1
TompaB
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Location: Sweden

Join Date: Jan 2014
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Default Amplicon sequencing of a really long single size 2.2 kb amplicon library

We have an ongoing study where we have designed a novel functional mapping approach using genetic barcoding of a large plasmid library. This is working really well and we have been sequencing many of them using MiSeq paired-end sequencing either 2x75 bp v3 or 2x150bp v2. (A large part has been to remove PCR chimerism during library preparation, but that we have solved now).

Now we have an interesting challenge! For one experiment we need to sequence the two ends (75bp is enough at each end) of a really long amplicon 2.2kb. This would be a very high diversity at both ends, but have a long strand of an identical sequence in between the ends (≈ 1.9kb). The entire library would have the a very narrow size distribution 2.1-2.4 kb.

I know that there are a number of previous attempts with long fragments presented here from a couple of years ago where short fragments are sequenced with a much larger abundance than the long ones, but have anyone tested the v3 chemistry with a single such long amplicon? Is it even possible? We can handle a decrease in cluster count. We can also run this on the NextSeq 500, potentially with the v2 chemistry (now suitable for amplicon sequencing if I have understood it correctly). As the clusters are much larger on the NextSeq flow cell, is this a better option for our really long amplicons?

Any tips are highly appreciated.

Last edited by TompaB; 02-22-2015 at 02:29 PM.
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