HiSeq V4 region with V2 chemistry

Theory behind mothur
1

I have just received the output from a Hi Seq run targeting the V4 region and using v2 sequencing chemistry (2 x 300bp).

It seems as though there are much more raw reads than when I have used Mi Seq runs.

In addition, when I checked the files with FastQC, the F reads are excellent, and the R reads pass the QC (>30) although there is some drop off after 250bp, so I will trim to 250bp before proceeding with the analysis.

I am trying this workaround in order to alleviate the difficulties associated with the v3 Mi Seq chemistry.

Two questions

  1. Should the higher numbers of raw reads be expected, given that this is a Hi Seq run?

  2. Can I use the Mothur Mi Seq protocol as usual with this Hi Seq data?

Thank you in advance

2

I suspect you’ll still have all of the problems you had with the V3 MiSeq chemistry. Why not just use the V2 2x250 MiSeq chemistry? One problem you’ll probably run into is that your 300 nt reads will probably extend beyond the length of the fragments you are sequencing. This is known (and admitted by Illumina) to increase the error rates.

Let us know how it goes…
Pat

3

Thanks Pat

I have trimmed the seqs to 250bp, but there seems to be a lot of raw seqs to work with.

Having said that the quality of the R read seems better than previous runs with the v3 chemistry.

I’ll see how it goes