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Old 03-15-2018, 05:32 AM   #1
minio.cz
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Default Running one sample with index primers?

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?
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Old 03-15-2018, 01:31 PM   #2
nucacidhunter
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That will work. You would have option of setting up run with index read or without.
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Old 03-16-2018, 09:27 AM   #3
thermophile
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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
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Old 03-16-2018, 12:22 PM   #4
minio.cz
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Thank you both. It should be obvious not to read indexes, but my brain is apparently not working lately
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Old 03-16-2018, 03:25 PM   #5
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Quote:
Originally Posted by thermophile View Post
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.

Last edited by nucacidhunter; 03-16-2018 at 03:29 PM.
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Old 03-19-2018, 06:27 AM   #6
thermophile
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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
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Old 03-19-2018, 09:16 AM   #7
minio.cz
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Quote:
Originally Posted by nucacidhunter View Post
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.
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Old 03-19-2018, 10:24 AM   #8
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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.
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Old 03-21-2018, 12:10 AM   #9
minio.cz
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Ok, final result. Turning off index read really works. Sequenced library without problem. In the end no PhiX has been added.
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Old 03-21-2018, 03:00 AM   #10
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No surprise since index reads are never part of the actual sequence in Illumina technology.
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