Troubleshooting demultiplexing issues using onboard DRAGEN on the NextSeq 1000/2000

DRAGEN software outputs two demultiplexing files called Demultiplex_Stats.csv and Top_Unknown_Barcodes.csv that can be used to troubleshoot demultiplexing issues. Where are the Demultiplex_Stats.csv and Top_Unknown_Barcodes.csv files located?

[runfolder]/Analysis/1/Reports

What information is in the Demultiplex_Stats.csv?

The Demultiplex_Stats.csv file provides the following information:

• sample ID

• index combination

• number of reads

• number of perfect index reads

• number of one mismatch index reads

• number of bases greater than or equal to Q30 (PF)

• mean quality score (PF)

  

What information is in Top_Unknown_Barcodes.csv?

The index and index2 columns provide the sequence of the unknown barcode as it was read by the instrument. The # Reads column provides the number of reads that the combination of index and index2 received.

When troubleshooting demultiplexing issues, use the index sequences identified for comparison to the expected index sequences (found in the sample sheet).

Possible causes for poor demultiplexing:

• Index sequences entered in the incorrect orientation in the sample sheet.

• Incorrect index sequences entered in the sample sheet (eg, Nextera vs TruSeq HT, or index A001 vs index A006).

• Poor Index Read sequencing quality (eg, index sequences containing lots of periods, representing “no calls,” near the top of the list).

For any feedback or questions regarding this article (Illumina Knowledge Article #3817), contact Illumina Technical Support [e-mail].