Question

Many 35nt reads with N bases in R1(paired-end) when sequencing with Illumina NextSeq

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5.1 years ago

mgdrnl ▴ 10

Dear All

We recently had a Quality Control issue when sequencing RNAseq with polyA capture in Illumina NextSeq. The reads were paired-end (2x75bp) and we found a proportion between 6%-9% of 35nt length reads with all N bases. It is also surprising that those reads all belong to the R1 reads of the paired-end library, the R2 reads look more or less ok.

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Anybody had the same experience and know the possible technical explanation?

RNA-Seq Qualitycontrol Illumina • 1.5k views

ADD COMMENT • link updated 4.7 years ago by Biostar 20 • written 5.1 years ago by mgdrnl ▴ 10

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Is there some adapter in your library ? If yes is it 35nt long ?

ADD REPLY • link 5.1 years ago by Nicolas Rosewick 10k

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As pointed above, the error could be a sample preparation, errors in base calling, reagents problems, ... Check with your provider and ask for a rerun of your samples

ADD REPLY • link 4.7 years ago by JC 13k

score 1 · Answer 1 · 2019-04-01

What did the sequencing facility have to say about this?

To me it seems like there was some sort of failure (software/hardware/reagents) with this run. Oddly the sequencer seems to have recovered from this failure half-way during read 1 (since you said read 2 looked ok).

Depending on the answer from the sequencing facility you would be within your rights to ask that they resequence the samples (if the problem was technical and not related to your libraries).