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Old 03-13-2018, 06:43 AM   #1
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Location: USA

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Default V4-V5 Region Using 2x250 - Enough Overlap?

Hi All,

We've been looking at the V4 region (515/806) in environmental samples for some time successfully. We'd like to move to the V4-V5 region using the 515/926 primers. That would result in a larger amplicon (~411 not including primer/barcode sequences).

Our sequencing provider runs 2x250 regularly but 2x300 only sporadically so the cost would be significantly more unless we have lots of samples. Does anybody have experience using 2x250 with this region? Will there be enough overlap of the paired reads to assemble?

Thanks for any insight!
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Old 03-13-2018, 11:24 AM   #2
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Location: Purdue University, West Lafayette, Indiana

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I think the critical factor is that at a cluster density where you produce the most sequence (even the most Q30 bases), the quality of single-plex amplicon runs deteriorates badly over the last 50 bases of a 250 cycle read. Whereas if you keep the density much lower--such that you may only generate 10 million or fewer clusters--the quality can be high all the way to the ends of the reads.

At these lower cluster densities we see pair merging occurring successfully at rate approaching 90% even with 450bp inserts.

600 cycle cassette? I don't know, is anyone getting reasonable 300 base reads with those?

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metagenomics, miseq 250bp

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