[thermophile]

I think personally, I'd rather have more confidence in my indexes, but I wonder if clients would freak out about loosing the last base of the read.

[ImmuneSeq]

Hey. The one time I had high undetermined content it was due to a wrong index primer in the experiment sheet. Software didnt find the index and thus put it all in the undetermined region. You can change the experiment sheet with the correct sheet and redo the demultiplexing.

But I would agree that ~10% can be okay for the 300 bp kit. Borderline though.

[fanli]

Hmmm…Interesting question and I hadn't considered the other limitation that may be coded into the MiSeq control software, no more that 525 cycles for v2, 500 cycle reagent cartridges which is maxed out with PE250, dual 8bp indexes

251 + 8 + (7)* + 8 + 251 = 525

(* 7 dark cycles before index 2 read.)

I'd rather keep the extra cycle at the end of the sequence reads in that case.

But you could be the first to publish a paper with 2x249 sequencing!

This would actually make a ton of sense for those of us doing amplicon sequencing where we build contigs anyways...that last base isn't super critical at all.