Miseq dual index Read1 (i7 index) problem?

[Min Jung Lee]

During miseq run, good quaility of read1 and read3 (i5), read4.
But read2 i7 index is low quality.
I already found many posting about read3 (i5 index) problem.
but I can’t find about i7 index failure.
I use Truseq HT dual index primer for 5 sample.
Help me please.

[nucacidhunter]

It could be that i7 indexes are not diverse enough. You can check Illumina pooling guidelines for duel indexed adapters (look at page 21).

http://sapac.support.illumina.com/co...5042173-01.pdf

[Min Jung Lee]

Thank you for your reply.
but, my library is enough diversity.

[nucacidhunter]

I am pointing to index diversity not the library. I wonder which wells from adapter plate you have used.

[pmiguel]

What sort of adapters are these that you are using?
Illumina instruments that prime i5 using the flow cell oligos (including the MiSeq) will always produce i5 sequence from a library molecule. But to produce an i7 read you need one of the i7 indexing primers to anneal in your amplicon. MiSeq's seem to have a fairly high annealing temp for their sequencing primers...

--
Phillip