High undetermined reads

[biancaneve0666]

Hi everyone, I have recently sequenced 1 lane of NovaSeq X Plus 10B with a sequencing provider. The library that was sequenced is a multiplex WGS library.

I received the result and was informed that I have high undetermined reads (approximately 56%) in my library.

They have also sent me the possible indexes for the undetermined reads and I was unable to find any of the sequences in my index list.

The index that I have used for the sequencing was used in the past as well and nothing of sort has happened before.

This is the %Pass filter vs %Occupied plot. The %Pass filter is in the 40-50% range, is there something wrong about this run?

Thank you for your kind help in advance!

[SnapSeq]

The %PF is low I think, but I don't think that this plot is necessarily the most helpful in resolving your issue. I haven't sequenced on an X Plus yet, but low %PF runs on the NextSeq 2000 and NovaSeq can still have good quality reads. What was the Q30 through each read, specifically the index reads? If the index reads had low quality, it's possible you could demultiplex with a higher mismatch allowance to cut down on the undetermined.

[robertsr]

Hello, replying to your post as I have found the similar results in some recent NovaSeq X Plus runs. My total undetermined reads were around 12% of my total reads and we demultiplexed the reads for every adapter that we have in our lab to ensure there was no contamination with unused adapters. Our Q30 scores average around 88% for each sample in the pool, the total PF% was ~60% and occupancy was ~93%. From what I can understand, a high number of undetermined reads can be expected. Hope that helps!