[error] No FASTQs matched your sample sheet’s lane, sample, and indexes. Please recheck your sample sheet

Question: My mkfastq pipeline crashed with "[error] No FASTQs matched your sample sheet’s lane, sample, and indexes. Please recheck your sample sheet". How can I troubleshoot this?

Answer: This error occurs during the MAKE_QC_SUMMARY stage of mkfastq, after bcl2fastq has completed in the BCL2FASTQ_WITH_SAMPLESHEET stage:

This error is usually triggered by one of three scenarios:

1. You were running an older version of bcl2fastq (2.17) on NovaSeq data. Please make sure you are using bcl2fastq 2.19 (2.20+ for the S4 flowcell).
2. All of the FASTQs ended up in Undetermined (something like Undetermined_S0_L001_R1_001.fastq.gz) because you either accidentally specified the wrong sample index sets in your sample sheet or there was a similar mixup of index sets in the lab. To test this hypothesis please see this article: What to do if I forgot which sample indices I used during library construction?
3. All of the FASTQs ended up in Undetermined because your sequencing run was low quality with a high error rate in the index reads, the BCL was corrupted, or other similar causes. To assess this hypothesis you would want to go over your Sequencing Analysis Viewer (SAV) files (RunInfo.xml, RunParameters.xml, and the Interop directory in your BCL directory).

Please contact [email protected] if you would like help troubleshooting this issue.