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Old 05-30-2016, 04:38 PM   #1
Pab77
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Default NextSeq - ribosome profiling weird results

Hi all,

We received a set of ribosome profiling libraries to be sequenced on the NextSeq 500. We ran the pooled libraries using 1 x 40bp chemistry on the NextSeq. The pool was qPCR quantified and run on the bioanalyzer. Please see the attached data. The clustering density was very broad across all 4 lanes. The % base plot showed a clear G signal across all 40 cycles! When fastQC was done, all 8 samples showed ~80% of the sequence to be G's! Since these were ribosome profiling libraries, we thought that most of the libraries could be rRNA and that resulted in low diversity that could be leading to inaccurate basecalling. Illumina told us that lower clustering, lower signal, and something library-related is causing this weird result. However, when the investigator ligated these libraries into a vector and sanger sequenced a few clones, all sequences were from genes and not rRNA. However, when these libraries were PCR amplified, no signal was detected at 14 cycles (protocol suggested maximum) but with 4 additional cycles, the correct library fragment size showed up. That suggested either very low template or very low PCR amplifiable molecules.

Could it be that higher number of PCR cycles introduced duplicates? Sanger sequencing shows genes and not rRNA. What could be the cause of these G's?
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Old 05-30-2016, 04:47 PM   #2
GenoMax
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NextSeq uses two color chemistry where an absence of signal is treated as a "G" base. Dr. Simon Andrews recently wrote a blog post that describes NextSeq chemistry and high confidence G signals.

There is something going on with these libraries and someone with experimental expertise may chime in with things to try. Perhaps there is a large fraction of fragments with damaged ends that are not able to cluster on flowcell.
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Old 05-31-2016, 04:30 AM   #3
Pab77
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Thanks GenoMax! The blog post was very helpful. Since the insert sequences seem to be fine for these libraries, could be adaptor related or damaged ends as you suggested. We've sequenced libraries generated from the same protocol some years back on the NextSeq without any problems, so the adaptor sequences should be fine. Thank you so much!
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