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Old 12-19-2018, 01:57 AM   #1
Location: Aarhus, Denmark

Join Date: Mar 2008
Posts: 52
Default NovaSeq index issue (high fraction of non-perfect indexes)

We have conducted a NovaSeq S1 run of different library types and noticed a lower %perfect reads from a single sample as compared to a MiSeq QC run of the same libraries.
The libraries were a mixture of 21x QIAseq FX, 9x KAPA RNA Hyper libraries (both with 8 bp dual indexes) and 36x KAPA Hyper DNA libraries prepared with NextFlex48 single indexes from BiooScientific (cat#514104).
We notice that a single KAPA Hyper DNA library with index sequence GTAGAGAT+TCTTTCCC (Barcode Adapter 17) were found to have 76 % perfect index match on the NovaSeq while it had 97.8 % perfect index match on the MiSeq. The insert sequences from the NovaSeq were furthermore much more noisy from this specific library.
All other KAPA Hyper DNA libraries performed fine on both MiSeq and NovaSeq.
The NextFlex48 indexes are single 6 bp index but can be read as 8 bp dual indexes resulting in an additional and universal terminal AT and an universal TCTTTCCC index 2 from all KAPA Hyper DNA libraries (see the attached pdf).

Wonder why a single library can behave so different on a MiSeq and NovaSeq- while similar libraries essentially only differing in the index sequence behave identical.
I have asked the same question to Illumina Tech Support and was asked to pass the question on to BiooScientific. I have done so, but wonder if any of you experienced NovaSeq users have encountered similar issues with specific indexes.
Best, /Jakob
Attached Files
File Type: pdf NextFlex48Adapters.pdf (10.6 KB, 18 views)

Last edited by JakobHedegaard; 01-15-2019 at 04:00 AM. Reason: Updating the header
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