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Old 02-28-2019, 06:50 AM   #1
Location: NYC

Join Date: Jun 2010
Posts: 18
Default Sequencing beyond the end of the fragment

Hi everyone

We've had a problem with a sequencing run and I would like to ask for a re-run with justification.

We submitted 4 libraries to size-select via pippin prep, pool and run SE 150 on a MiSeq. The pippin prep was to select between 200-600bp fragments, and apparently worked well (image of one of the libraries attached, the others looked the same).

The run came back and it's pretty obvious that something went wrong. between 70-80% of the reads are primer dimers, sized 135bp. I'm attaching the FastQC per-base quality. The first 6bp were in-line barcode. After that there is a big drop in quality at the 77bp mark, which is the end of the primer dimer fragment. However, the fragments keep giving base calls, albeit at very poor quality. Is this what the software does when the reads go beyond the end of the fragment?

The discrepancy between the reads and the bioanalyzer profile post-pippinprep makes me think that they messed up and loaded the pre-pippinprep sample into the flow cell. But I can also imagine that the bioanalyzer could give a wrong size profile in some conditions, or that there could be some primer dimer contamination that would not be visible on bioanalyzer.

Does anyone have experience in this kind of problem? Should the read lengths from the MiSeq run be concordant with the bioanalyzer profile, or is that too much to ask?

Thank you in advance!
Attached Images
File Type: png BApostpippin.png (97.2 KB, 19 views)
File Type: png fastQCpbq.png (39.9 KB, 16 views)

Last edited by zaratieg; 02-28-2019 at 07:01 AM.
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Old 02-28-2019, 07:27 AM   #2
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Location: Florida

Join Date: Apr 2018
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The trace that you are presenting here, shows fragments that go beyond 600 bp that are even overlapping with the upper marker. Is this picture from the library before size-selection? That's what it looks like.
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Old 02-28-2019, 07:47 AM   #3
Location: NYC

Join Date: Jun 2010
Posts: 18

Thanks for your reply. No, this is supposed to be post size selection. I didn't pay attention to that part, I thought that was their size marker. I saw the well defined bump between 200-600 and assumed that that's what was selected. Does that mean that the pippin prep purification failed?
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bioanaly, miseq, pippin

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