V4-V5 Region Using 2x250 - Enough Overlap?

nsharma · 03-13-2018, 06:43 AM

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!

pmiguel · 03-13-2018, 11:24 AM

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?

--
Phillip

nsharma · 03-25-2018, 05:30 AM

Sorry for the late reply. Thanks! This helps a lot. We're discussing with our service provider and will be doing some test runs to evaluate the data.

thermophile · 03-26-2018, 05:35 AM

I wouldn't move to the longer region on the 2x250 kit. Allegedly the 2x300 is better in the past ~year but I haven't tested it out so continue offering amplicons that are <300bp long.

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03-13-2018, 06:43 AM	#1
nsharma Junior Member Location: USA Join Date: Mar 2018 Posts: 2	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!

03-26-2018, 05:35 AM	#4
thermophile Senior Member Location: CT Join Date: Apr 2015 Posts: 243	I wouldn't move to the longer region on the 2x250 kit. Allegedly the 2x300 is better in the past ~year but I haven't tested it out so continue offering amplicons that are <300bp long. __________________ Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

03-13-2018, 11:24 AM	#2
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,318	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? -- Phillip

03-25-2018, 05:30 AM	#3
nsharma Junior Member Location: USA Join Date: Mar 2018 Posts: 2	Sorry for the late reply. Thanks! This helps a lot. We're discussing with our service provider and will be doing some test runs to evaluate the data.