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NovaSeq Xplus top unknown barcodes

I'm adding small subset of lane wise data.

Lane Index Index2 # Reads

1   TCCTCATGAT  GGGGGGGGGG  6,084,098
1   AGCGTACGTC  GGGGGGGGGG  6,019,020
1   CCTAACGTAA  GGGGGGGGGG  5,337,515
1   TTGCGAGAAT  GGGGGGGGGG  4,880,048
8   GGGGGGGGGG  GAATCGTGGT  28,217
8   GGGGGGGGGG  ACATCAGTAG  28,197
8   GGGGGGGGGG  GTTATGGAGT  28,159

I could see from the resources https://knowledge.illumina.com/software/cloud-software/software-cloud-software-reference_material-list/000002875

Based on the above resources

For sequencing systems using Illumina one-channel (iSeq) or two-channel SBS chemistry (MiniSeq, NextSeq 500/550 and NovaSeq), poly-G sequences indicate that no index sequence was read. Poly-G sequences are typical for PhiX reads, which are not indexed.

One similar query what cause poly-G from NextSeq

What other factors can be attributed to this sort of poly G in the index that is part of these top Unknown barcodes

Any suggestion or resources would be helpful

fastq

Does this run have single and double-indexed libraries pooled together?

1 answer

Considering this is a XPlus run this set of reads must be a small fraction of the total output (which we can assume was fine?).

If this is how majority of the data looks then the best option is to get in touch with Illumina support and see if they can look at the entire metrics/instrument remotely and help diagnose the issue.

Stuff like this happens at times and can't always be logically explained. If this is a minor portion of the data then set the data aside and move forward with the rest.

Note 1: As @Arup says, if this is a run with mixed 1D/2D indexes then this would be a normal result. Demux may need to be run separately for the two sets of indexes.

Note 2: Note 1 may not explain the second set of example indexes posted above. That seems to be partially failed index 1 which Illumina support should be able to help diagnose.

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