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JakobHedegaard 12-19-2018 01:57 AM

NovaSeq index issue (high fraction of non-perfect indexes)
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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

JakobHedegaard 12-20-2018 12:24 AM

Come on - I have heard several rumors about indexes behaving non-optimal on the NovaSeq. Please share your experiences :-)

GenoMax 12-20-2018 04:02 AM

MiSeq is a sequencing champ as far as illumina libraries are concerned. We look at results from MiSeq nano runs only qualitatively (rather than quantitatively) before the pools are transferred for final NovaSeq runs. You always need to keep in mind G = no signal limitation of 2-color chemistry as well.

If you are consistently observing that index combination performing badly then BioO would need to know that.

pmiguel 12-20-2018 11:40 AM

You are running non-UDI indexes on the NovaSeq? Are you not concerned about index swapping?

We demultiplex allowing 0 mismatches for all Illumina runs these days. So I haven't taken note of non-concordant perfect index matches between the MiSeq and NovaSeq.

However, this is an issue mentioned in the Illumina White Paper on using an iSeq to titrate reactions for a NovaSeq.

luc 12-29-2018 12:39 PM

I would suggest to check the index reads for this particular library to see which base is affected.

JakobHedegaard 01-15-2019 04:03 AM

I have been told that two TruSeq UD indexes have shown lower than expected perfect index reads: UDI0025 (ACTAAGAT, AACCGCGG) and UDI0055 (AGGCAGAG, AGAATGCC).
Anyone else seen this?

JakobHedegaard 01-15-2019 04:12 AM

And in a recent S2 (2x150) NovaSeq run of eight Nextera DNA Flex libraries prepared from human gDNA using indexes from the CD Indexes (96 Indexes, 96 Samples, cat# 20018708) we observed a single library with only 24-27% perfect reads. The index was from well F04, H701 (TAAGGCGA) + H522 (TTATGCGA) and the top non-perfect index was TAAGGCGA+TTTTGCGA (so a T in pos 3 in index2 where it should have been a A).
The other indexes in the run were find with a high fraction of perfect indexes.
Other observations of indexes with high fraction of non-perfect indexes?
We allow a single mismatch so we do get the data we need. We are though a bit worried that the quality may be affected.
And it would be great with an explanation of this index issue on the NovaSeq!

Pichelsberg 01-15-2019 03:32 PM

Helpful thread.

JakobHedegaard 01-18-2019 01:18 AM

In our hands, we do not find any significant differences in the quality between insert data with perfect index reads and insert data with 1-mismatch index reads.
So despite a much larger fraction of 1-mismatch index reads the data is fine. Nice...

But it would be great with an explanation of how/why this index issue on the NovaSeq actually happen.
In our data, the issue is more frequent in index2 than in index1.
Can it have something to do with a more "loose" binding to the surface oligo when priming for reading index2? Or a sequence-specific higher (and systematic) error rate in index2 reading?
Any ideas?

And a list of indexes where one should expect a higher fraction of non-perfect indexes would be nice. Should we start one?

ralyma 01-25-2019 06:15 AM

Dear Jakob,

We also have observed problems with H522=TTATGCGA. In demultiplexed data (default settings accepting 1-mismatch) 97% of observed adapters are TTTTGCGA.

To my knowledge TTTTGCGA is not an Illumina adaptor, so this makes it less likely that we have mixed H522 with another adaptor. Then it is more likely that there has been has been an issue in the production of H522 so that sequence TTTTGCGA has been produced instead of TTATGCGA.

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