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Old 10-13-2018, 09:22 AM   #1
JBKri
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Default Share run between NEBNext smallRNA and 16s amplicons in v3-600 MiSeq run?

Hi all,
for testing purposes we are thinking to put a small amount (~2-3 %) of a smallRNA library (NEBNext) in a run that will otherwise consists only of 16s amplicons (~490 bp, Nextera-type 8 bp dual indexes). Would this be likely to cause any problems? Obviously the size of the amplicons is much bigger, so the clustering efficiency might be very different, but by adding such a small amount I don't think it should be a problem. I looks to me like the NEBNext kit uses the TruSeq sequencing and index primers, so we would get both a forward and reverse read of the smallRNA, along with a "fake" index i5.
Edit: Correction, the index read (i7) and reverse read would be using the TruSeq primers, but for the forward miRNA read it would use another primer; I can't figure out which from the information available from Illumina. I suppose it is in there in the HP10 primer mix.

If everything looks OK from the testrun, the plan is to do a v3-600 run of the smallRNA library, but with a long since expired sequencing kit, and using less than the available 600 cycles. We hope the quality will be sufficient for the few cycles we need even from a very much expired kit.

Last edited by JBKri; 10-15-2018 at 02:37 AM. Reason: corrections
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Old 10-13-2018, 11:05 AM   #2
luc
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As long as the short library molecules are a smaller fraction of the loaded libraries (<20%?) there will be no problem.
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Old 10-16-2018, 01:04 PM   #3
pmiguel
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I agree, it will probably work fine for you.
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Phillip
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