Troubleshooting multiple entries with identical indices error during run setup on NovaSeq X Series

During run setup for a NovaSeq X/X-Plus run, users may encounter an error stating "Multiple entries with identical indices XXXXXX and XXXXXXX in Lane #" for a single or multiple indexes or index pairs, as shown below.

This error may indicate a single index/index pair or may list a large number of identical indexes. Users will not be able to proceed with run setup until the duplicate indexes are removed. There are multiple possible issues that may cause this error. Below list possible causes of this error and the required steps for resolution.

Possible cause 1: An index sequence has been entered for multiple samples in a given lane.

To determine if this has occurred, users can search for the indicated index sequence in their sample sheet and determine if the given index or index pair is present in the lane indicated by the error. If multiple instances of the index/index pair are present, the index must be changed to a unique index/index pair. Only samples that are uniquely indexed within a run will be able to be demultiplexed.

Possible cause 2: Upload of a Sample Sheet with incorrect formatting.

If users are attempting to upload a sample sheet into BaseSpace run planning to populate indexes, it is possible that use of a sample sheet with incorrect formatting may result in a similar error. In order to determine if this is the case, verify that the sample sheet uploaded contains the following columns.

Lanes | Sample ID | Index 1 | Index 2 (if applicable) | Barcode Mismatches Index 1 | Barcode Mismatches Index 2 (if applicable) | Project

All the above headers must be present within the sample sheet for BaseSpace to correctly interpret and assign indexes to samples appropriately. Additionally, verify that indexes are correctly entered into the sample sheet and that if a sample is to be run in multiple lanes that the appropriate lanes are indicated in the "Lanes" column with the format #,#,#... with no spaces present.

For any feedback or questions regarding this article (Illumina Knowledge Article #9153), contact Illumina Technical Support [e-mail].