# 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)

  ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-78915941808e142f20188ea22a976522617d53ec%2Fimage1.png?alt=media\&token=d94edfe9-a8f0-4a46-b395-bc9833122337)

**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.

![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-0619a9d7419c1626713ff671c624cb238801dbc4%2Fimage2.png?alt=media\&token=f11c3106-2dfc-4ef5-a80c-360ce1e66993)

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).

\
\
\ <br>

|                                                                                                                                                                                                                                                                                                                                                                 |
| :-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: |
| *For any feedback or questions regarding this article (Illumina Knowledge Article #3817), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000003817%20-%20Software%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |