Running one sample with index primers?

[minio.cz]

Hi all,
I have a question - I am going to prepare library and I want try it with single sample and do a test run on MiSeq. The poblem is that I have just NEBNext Multiplex Oligos which are supposed to be multiplexed. Will the sequencing work if I add PhiX?

[nucacidhunter]

That will work. You would have option of setting up run with index read or without.

[thermophile]

Only one index could cause the run to fail during the index sequencing because there isn't any diversity in each cycle. I'd just set up the sample sheet with 0 index

[minio.cz]

Thank you both. It should be obvious not to read indexes, but my brain is apparently not working lately

[nucacidhunter]

Only one index could cause the run to fail during the index sequencing because there isn't any diversity in each cycle. I'd just set up the sample sheet with 0 index

A single index sequencing on HiSeq systems is fine and on average 98% of reads correctly demultiplex. I wonder if you have seen single index sequencing fail on MiSeq or speculating.

Edit: If PhiX added to run, sequencing with index read reduces follow up work required for removing PhiX reads in some workflows.

[thermophile]

Yes I've had runs with 50% phiX fail during indexing on the MiSeq-I'd guess something similar would happen if you only use one index (3/4 colors would be dark and the machine would likely throw the "focus out of range" error). OP said they were going to run this on the miseq

[minio.cz]

A single index sequencing on HiSeq systems is fine and on average 98% of reads correctly demultiplex. I wonder if you have seen single index sequencing fail on MiSeq or speculating.

I am speculating. I am testing library prep and would like to test library first on MiSeq if it contains what I want. But have only multiplexing oligos.

[GenoMax]

If you underload the library deliberately the run should be able to complete even with just one index. I am reasonably sure we have done this in the past but will have to confirm.

[minio.cz]

Ok, final result. Turning off index read really works. Sequenced library without problem. In the end no PhiX has been added.

[GenoMax]

No surprise since index reads are never part of the actual sequence in Illumina technology.