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Old 10-03-2017, 02:16 PM   #1
jreuther
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Default Illumina MiSeq Poor Q30 scores in Read 1 relative to Read 2

Hi everyone,
We are consistently seeing an issue on our MiSeq where Read 1 has very poor Q30 scores, followed by a successful sequencing of Read 2 with Q30 scores above 80%. We have had successful runs where we didn't see this trend but it seems about every other run we see this happening. We have talked with Illumina Tech Support and they don't seem to know what is going on. They adjusted the temperature of the instrument (set it a few degrees lower) but this didn't seem to fix the issue. Has anyone else had similar issues? Any insight would be much appreciated.
Thanks! See below for run specifics:


Chemistry: Miseq v3 600 cycle kit.
Read configurations: R1 = 150 cycles, Index = 16 cycles, R2 = 150 cycles.

The library is a DNA hybridization capture, with avg library length ~320 bases

The library was run with 10% PhiX spike-in.

Custom primers were NOT used.
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Old 10-03-2017, 06:30 PM   #2
luc
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Indeed very weird. Fortunately we have not seen this problem so far.
Are you using custom sequencing primers?
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Old 10-03-2017, 10:18 PM   #3
nucacidhunter
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Two possible causes:
1- Instrument related
2- Library P5 adapter

In case2 the adapter sequences could have variations from standard sequences which affect R1 primer annealing or binding stability resulting in low quality. Paying attention to phasing/prephasing in good and bad runs should highlight some differences. This case is more prevalent in PCR amplicon based library preps that adapters are added by PCR because of the possibility that some oligos will have indels or base substitutions resulting from low quality synthesis or storage.
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Old 10-24-2017, 04:39 PM   #4
jlli2000
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Does anyone have experience to run Miseq V3 600 kit with uneven r1 and r2 cycles? i.e. r1 400 and r2 200?

Thanks,

Jin
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