Ilumina V2 vs V3 for amplicon sequencing

Question

[joelslade]

Hi there,

I am having a dilemma -- I am using an Illumina MiSeq to sequence MHC in songbirds (which can have many alleles per individual). I was able to create a primer to get sequences that range from 370-420 bp (due to indels and differences between subspecies). This means some sequences will not have substantial overlap if I use V2. In the past I have used V3, but my new genomics core seems to not want to use V3 due to its issues (which is still don't fully understand).

I am trying to determine what is more risky, having only 80 bp of overlap if I use V2 or having more overlap, but experiencing some issues (again which I don't understand) using V3.

Any advice is appreciated.

[h.mon]

Illumina released the MiSeq V3 kits and claimed they could be used to obtain 2x300bp. However, very often when using this setting, the quality of the reads is terrible, and they will fail to overlap. Using V3 with 2x250 reads, or V2, seems a safer choice.

[joelslade]

Thanks so much for your response.

I must be confused by the terminology. I just assumed that V3 meant 2 x 300 bp, but does it just mean you can only use V3 to do 2 x 300 bp, while V2 is reserved for the lower read lengths? I assume V2 2 x 250 bp is the most robust?

[h.mon]

Please use the ADD REPLY button when commenting on someones answer or comment.

I don't remember dealing with MiSeq V2 data, so I can't compare it to V3, but I think for 2x250 reads, the quality of both are similar. However, Illumina MiSeq specifications state you should get a lot more reads from V3 kits: for paired end reads, MiSeq V2 should yield between 24–30 million reads, while MiSeq V3 should yield 44–50 million reads.