MiSeq v2 cartridges for metagenomics

[Allison Harmon]

Hi all,
Has anyone used the MiSeq v2 cartridges for 16s metagenomics? Illumina recommends using the v3 cartridges for better metrics. I have plenty of v2 on hand that will like to use for metagenomics. Thanks

[Brian Bushnell]

You obviously can't get full-length 16S with either of them, but you can get full-length V4 region with both of them (if you run pair-ended and merge the reads with a tool such as BBMerge). So, I don't think there's a significant disadvantage to using your v2 kits unless it has some kind of biases of which I am unaware (which is unlikely).

[Allison Harmon]

I am looking to amplify regions v3 and v4 following the Illumina protocol.
It is about 460bp

[Brian Bushnell]

Hmmm... if you merge reads, the max for 2x250bp is slightly under 490bp, while the max for 2x300bp is slightly under 590bp. 460bp sequences would be fine either way, but as you get closer to the limit, the results will become biased against longer sequences, and ultimately 500bp sequences would not be merged at all from 2x250bp reads. So it's important to precisely characterize the length distribution of your region of interest before selecting a method. As long as ~99% of the known sequences are shorter than 490bp, 2x250bp reads should be fine for most purposes.

[kmcarr]

In our experience 16S amplicons sequence very, very poorly with the version 3 chemistry. The quality drop off in the 3' ends of the reads is so extreme that after trimming you may as well just have started out with PE250 version 2 reads. We regularly sequence both V4 and V3-V4 amplicons using version 2 PE250 reads.