Hi BioStar community,
We have been routinely sequencing DNA samples using an Illumina MiSeq sequencer with CDI QIASeq Fx library kit. So far, we have not encountered any issues, as the MiSeq performed the demultiplexing process itself. However, in a recent run, the MiSeq failed to produce any fastq files except for the undetermined R1 and R2 reads.
To address this problem, we attempted to demultiplex the run ourselves using bcl2fastq2, but unfortunately, we encountered the same issue. Upon inspecting the Demultiplexing Stats, we observed that the I5_Index contains multiple Ns instead of the expected 8 bp sequence.
Most Popular Unknown Index Sequences Columns: Index_Sequence Hit_Count ATTACTCG+TNNNNNNN 59644 TCCGGAGA+TNNNNNNN 58629 CGCTCATT+TNNNNNNN 58571 ATTACTCG+ANNNNNNN 52028 TCCGGAGA+ANNNNNNN 51507 CGCTCATT+ANNNNNNN 49504 ATTACTCG+CNNNNNNN 44697 ATTACTCG+GNNNNNNN 41469 CGCTCATT+GNNNNNNN 41314 CGCTCATT+CNNNNNNN 40904 TCCGGAGA+CNNNNNNN 37623 TCCGGAGA+GNNNNNNN 36295
In this sequencing run, we used a combination of 701-703 for I7 and 501-506 indexes to assign 24 samples. Consequently, both indexes are required for successful demultiplexing. Because of this, we have come to the realization that this issue might not be recoverable from a bioinformatics point of view.
We are puzzled as to what could have caused this problem. Could you please share any insights or suggestions on potential causes for the unexpected Ns in the I5_Index during demultiplexing? The sequencer seems to have completed all cycles without issue, there were no errors or warnings reported and all 518 bcl files seem to be there.
You can find attached the flow cell & lane summaries:
You can also find the sample sheet used:
Thank you in advance for your assistance!