Paired end: 2x151 vs 2x250 for V4

[Mike_McFarlin]

Hi everyone,

I am about to send samples of extracted DNA out to a lab for library preparation and sequencing. I will be using the 515f-806r primers for the V4 region. I am unsure whether I should use 2x151 or 2x250 paired end sequencing for this region.

The sequencing strategy used by the lab I am working with does not include the primers in the reads. From a previous post it seems like the 2x151 sequencing will be sufficient to merge reads if the primers aren’t included in sequencing but I wanted to double check since this work is quite new to me.

Thank you for your help,

Mike

[sixvable]

Hi @Mike_McFarlin

Always choose PE250 mode for amplicon sequencing.

Sincerely,
sixvable

[Nicholas_Bokulich]

Hi @Mike_McFarlin,
Welcome to the forum community!

@Mike_McFarlin

I will be using the 515f-806r primers for the V4 region. I am unsure whether I should use 2x151 or 2x250 paired end sequencing for this region.

As @sixvable pointed out, 2x250 would be best. 2x151 barely gives enough overlap to cover the 515f-806r amplicon reliably… especially if there is any loss of quality at the 3’ ends of the reads. This is a very common issue, and commonly dooms 2x151 V4 runs to be utilized as single-end data.

So 2x250 is a little more expensive, but less risky than 2x151.

Good luck!

[SoilRotifer (Mike Robeson)]

Hi all, the decision of whether or not to use 2x150 or 2x250 depends on your sequencing protocol.

For more details on this see the following post:

Trimming and joining short reads resulted in few merged read on v4 region

Hi @simon, If you are indeed sequencing via a 2x150 kit with the EMP V4 (515f-806r primers), and those primers are actually contained within the reads… then sadly these reads will either be to short, or not have enough enough high-quality bases, for merging. The 2x150 sequencing approach works best when using the sequencing strategy outlined here, and the references therein. That is, when using a 2x150 for the V4 region, the sequencing strategy used should NOT sequence through the primers. Mea…

It looks like the EMP web site is not currently accessible, so checkout the protocols.io version.

[Mike_McFarlin]

Hi everyone,

Thank you so much for the responses.

@SoilRotifer, since the sequencing that I will be using does not sequence through the primers, as stated in your previous post you shared, it seems like it would be safe to use 2x150. Am I reading that correctly?

Thank you,

Mike

[SoilRotifer (Mike Robeson)]

Hi @Mike_McFarlin,

Yep! Many of the data sets I’ve worked with are from Argonne National Lab, which consists of output generated from their 2x150 V4 EMP protocol. I’ve not had trouble merging the paired end output from these so far.

In fact, here is a data set from a small pilot project my colleagues and I performed using this approach.

-Cheers!
-Mike

[Mike_McFarlin]

Hi @SoilRotifer,

Thanks, this is a huge help! I’ll be working with Argonne National Lab for the library prep and sequencing so it’s nice to hear you’ve had no issues merging paired end from 2x150.

-Mike