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Old 09-01-2020, 01:51 AM   #1
aasako
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Default Problems sequencing a low diversity library on NextSeq

Hi everyone,

Weíve done several attempts at sequencing a low diversity library on a NextSeq Mid Output v2.5 flow cell, following the recommendations from the provider of the library preparation kit. Unfortunately all samples failed QC for low coverage. We noticed a few things that we havenít been able to explain:

1. 30% PhiX was added but we only got out ~5%. We have done several attempts at sequencing this kind of library, adding between 20-40% PhiX, but measured PhiX is always considerably lower than added. PhiX from the same batch was used before and after our run(s) with little discrepancy between added and measured PhiX.
2. The thumbnail image shows normal cluster density but when it comes to clusters that were read thereís a large difference between the top and the bottom of the flow cell with very few clusters read at the bottom.
3. There is a drop in quality around cycle 20-40.

For the last run the flow cell was loaded with 1.5 pM, cluster density was 133 k/mm2 and the error rate <1%. We have checked for primer dimer contamination in the library pool but did not find this. There was a discrepancy between the concentration measured with Qubit and qPCR (almost twice as high concentration with Qubit), but this was the same for the PhiX and the library pool, so it doesnít explain low measured PhiX. The input was based on Qubit, which makes overloading of the flow cell unlikely. Weíve been in contact with Illumina tech support but havenít managed to find an explanation to why things went wrong, and therefore also donít know what adjustments to make when we resequence.

Any suggestions/ advice would be very much appreciated!

Thanks.
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Old 09-01-2020, 12:19 PM   #2
itstrieu
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What are you libraries size? I believe PhiX is around 500 bp so smaller libraries will bind more efficiency throwing off your PhiX alignment.

Also we ran a couple of mid output kits and noticed an issue with the flow cell where cluster of tiles produced high error rate.
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Old 09-02-2020, 12:49 AM   #3
aasako
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Thank you for your input. The library size is about 150 bp, so it's smaller. We thought about this as a possible explanation to why PhiX wouldn't align properly. However, some time ago one of my colleagues sequenced libraries prepared with the same kit and did not encounter this problem. I've also asked the provider of the library preparation kit, and they haven't seen this before.

For some reason I can't open your attached picture, so I only get to see a miniature of it, but there seems to be less of a pattern than it was on our flow cell.
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Old 09-02-2020, 04:07 AM   #4
GenoMax
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Have you tried to deliberately underload the flowcell? Other option is to try sequencing on MiSeq, undeniable champ of hard to sequence libraries.
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Old 09-02-2020, 06:06 AM   #5
aasako
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I don't think it is underloaded (though it would probably be possible to load a bit more). The problem seems to be that the clusters are not read. At the inlet of the flow cell the cluster density is about 170 k/mm2 and PF 92%. It is then gradually decreasing to 122 k/mm2 and 0% PF.

The MiSeq could definitely be an option, but as it has lower capacity we were hoping that we could make it work on NextSeq.
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Old 09-03-2020, 10:11 AM   #6
cmbetts
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You implied that you used a specific library prep kit and are following manufacturer's recommendations. Can you share which kit so we could better understand potential library structure issues?
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Old 09-04-2020, 12:44 AM   #7
aasako
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We are sequencing T cell receptors and the libraries were prepared with the immunoSEQ hTCRB Kit from Adaptive Biotechnologies.

I've now found out that the region where there is a drop in quality (20-40bp) is the index primers. If PhiX had aligned to the same extent as added, I suppose the low diversity in this region wouldn't have been a problem.
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Old 09-08-2020, 06:41 AM   #8
itstrieu
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The first 25 cycles or so is the most important for library diversity. Was the Phix made fresh for this run or was it from an aliquot? I sometime refreeze denature Phix for later runs and after awhile, the alignment would be off.
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Old 09-17-2020, 06:02 AM   #9
aasako
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I'm not sure if the PhiX was made fresh for this run, as I didn't perform the sequencing myself. However, the problem with less aligned PhiX than added has been a recurrent one and only with our libraries.
We have now resequenced the samples and this time it went well. Though we still didn't get out as much PhiX as added we got more than last time, and the PF was fine.

Thank you for all input/ suggestions!
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