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Old 11-08-2016, 05:34 PM   #1
incognitos
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Default Miseq v3 600 cycle kit - poor read 1 quality

Hi all,

I recently completed a run using the Miseq v3 600 cycle kit.
The read configurations were as follows:
R1 = 300 cycles, R2 = 55 cycles, Index1 = 6 cycles.

The library is an amplicon style construct of avg length ~550 bases and has a constant sequence at the beginning of Read 1 (~60-65 bases).

The library was run with 20% PhiX spike-in. Custom primers were used, but these have been successfully used with v2 miseq kits as well as Nextseq kits without issue.

Essentially, I need at least 250 bases on Read 1, whereas Read 2 need only be a max of 50-55 (no need for overlap on Read 1 and 2).

I tried the v3 kit but the FastQC files show that the run was not particularly great for read 1. See attached slide for one of the libraries multiplexed in this run. Reads drop below Q30 pretty much within 100 bases.

Does anyone have any insight here? After perusing through previous posts from a while back, I was thinking of maybe switching to the Miseq v2 300 cycle kit or the 500 kit. Do you think this is a good idea (e.g. going with long read for read 1 with 300 cycle kit)? Previous threads suggest that 500 kits have also been affected although their quality is still better than the 600 cycle kit.

Appreciate your feedback!
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File Type: ppt Fastqc.ppt (121.0 KB, 26 views)
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Old 11-09-2016, 05:44 AM   #2
thermophile
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Why pay for a 600 cycle kit but not use all of it?

The qscores drop faster for v3 kits than v2. Regardless which kit, run the full thing so you have overlap to error correct the poor quality bases
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Old 11-09-2016, 05:56 AM   #3
incognitos
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Yes I see the waste of the reagents, but the 3'end of the library has a long stretch of Ts due to the polyA signal from mRNA. So going beyond 50 bases in the 3'end will result in unusable sequence anyways.

I was wondering if anyone has any experience doing longer than 150 bases for read 1 with the miseq v2 300 cycle kit? I was considering an asymmetric run of 250 and 50 for R1 and R2, respectively.
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Old 11-09-2016, 09:46 AM   #4
microgirl123
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There's no reason you can't do 250x50 with a 300-cycle kit. You should end up with similar Q scores to Read 1 on a 500-cycle kit, which are okay at the end (not great, but okay).
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