NextSeq Data Shifted by One Line During Demultiplexing — What Could Cause This?

Question

[pkk]

I recently encountered an issue where, during the demultiplexing process of data generated by a NextSeq run, the data for samples appears to be shifted by one line. Here are the conditions:

The sample sheet, index barcodes, and the required settings for demultiplexing have not been changed. However, the data generated during this run seems to have shifted for each sample by one line. I’m curious if anyone has experienced similar issues and what could cause this to happen. Could this be related to a sequencing machine calibration issue, or perhaps something in the base calling or indexing process? Any suggestions for troubleshooting would be appreciated.

[GenoMax]

Can you add some clarification as to what you mean by "shifted by one line"? Are you saying that samples got the wrong indexes e.g. sample 2 from sample sheet got indexes for sample 1?

This may simply be an issue with the samplesheet used for demultiplexing.

[pkk]

To clarify, by "shifted by one line," I mean that the sequencing data for a given sample appears to be misaligned by one position. For example, the reads that should belong to sample 1 are instead showing up under sample 2, and so on. This is happening consistently across all samples, not just for a single sample or index.

I have already double-checked the sample sheet and index barcodes, and they are correct. The sample sheet hasn’t been changed, and it worked fine in previous runs. So, I don't believe the issue lies with the sample sheet.

Could this type of issue be related to the sequencing machine itself or perhaps a problem during the bcl to fastq conversion? I'd appreciate any further insights!

[pkk]

Actual Index Information Used:

Below is the table showing the i7 and i5 index barcodes that were used in my sequencing run. Each sample is associated with specific indices. Despite this setup, I encountered an issue where the sequencing data appears to be shifted by one line during the demultiplexing process.

 I7_Index_ID            index               I5_Index_ID          index2

1. CAAAAGNNNN CAAAAGNNNN NNNNNNNNNN NNNNNNNNNN
2. CAACTANNNN CAACTANNNN NNNNNNNNNN NNNNNNNNNN
3. CACGATNNNN CACGATNNNN NNNNNNNNNN NNNNNNNNNN
4. CACTCANNNN CACTCANNNN NNNNNNNNNN NNNNNNNNNN
5. CAGGCGNNNN CAGGCGNNNN NNNNNNNNNN NNNNNNNNNN
6. CATGGCNNNN CATGGCNNNN NNNNNNNNNN NNNNNNNNNN
7. ACTTCTGCNN ACTTCTGCNN CCGGGACTNN CCGGGACTNN
8. TTAAGCAGNN TTAAGCAGNN GTCCAGGANN GTCCAGGANN
9. ATCAAATCNN ATCAAATCNN TCATGTAGNN TCATGTAGNN
10. TTTGAGTCNN TTTGAGTCNN CTATCCGGNN CTATCCGGNN
11. AAATCCTCNN AAATCCTCNN GGCGGGTTNN GGCGGGTTNN
12. TACAGATGNN TACAGATGNN CACGTTCGNN CACGTTCGNN
13. TAAGCGCANN TAAGCGCANN TTCTACGGNN TTCTACGGNN
14. CAACGGAANN CAACGGAANN AGTAGATGNN AGTAGATGNN
15. GCCTCTATNN GCCTCTATNN AAATCAGCNN AAATCAGCNN
16. TTAATAGCAC TTAATAGCAC CAGACGTGGT CAGACGTGGT
17. CATTCACGCT CATTCACGCT TGAGTACAAC TGAGTACAAC
18. GGCACTAAGG GGCACTAAGG GTTGACCTGA GTTGACCTGA
19. ATTCGGTACA ATTCGGTACA TCCTCGGACT TCCTCGGACT
20. CGGCTAATNN CGGCTAATNN AGAACGAGNN AGAACGAGNN
21. ATCGATCGNN ATCGATCGNN TGCTTCCANN TGCTTCCANN
22. GCAAGATCNN GCAAGATCNN CTTCGACTNN CTTCGACTNN
23. GCTATCCTNN GCTATCCTNN CACCTGTTNN CACCTGTTNN
24. TACGCTACNN TACGCTACNN ATCACACGNN ATCACACGNN
25. TGGACTCTNN TGGACTCTNN CCGTAAGANN CCGTAAGANN
26. AGAGTAGCNN AGAGTAGCNN TACGCCTTNN TACGCCTTNN
27. ATCCAGAGNN ATCCAGAGNN CGACGTTANN CGACGTTANN

Note that the issue only occurred between samples 7 and 15.

If anyone has insights into why this might be happening or suggestions on how to troubleshoot it, I’d greatly appreciate your input!

[GenoMax]

Why do you have 4 indexes per line? I7/I5_Index_ID is generally a code like N701 etc.

Are you sure this kind of samplesheet has worked in the past? You should carefully check the demultiplexing report and the "top unknown index" report.

[Arup Ghosh]

If the libraries were prepared using Illumina kits, use the Illumina experiment manager to prepare the sample sheet to avoid any user errors.