# Best Practices for Pooling Libraries on NovaSeq X Series to Mitigate Lane to Lane Contamination

**Background**

The NovaSeq X Series systems introduced individually addressable flow cell lanes with automated onboard independent lane loading. This was achieved by reducing shared fluidics lines and isolating sample loading and cluster-generation chemistry steps between lanes. When operating as intended, automated lane loading is designed to minimize any risk of lane-to-lane contamination.

However, rare situations have been observed where contamination has occurred. To help reduce occurrence and mitigate the impact of this contamination, we recommend following the best practices below regarding sample indexing.

**Best Indexing Practices to Reduce Impact of Lane-to-Lane Contamination**

1. Use index reads to demultiplex samples during every run.
   1. If you are sequencing only a single sample in a lane, include Indexed PhiX and use indexing cycles to help identify any lane-to-lane contamination.
   2. See Knowledge Article [PhiX Indexed Control (1000 Cycle) Product Information](/instrumentation/general/instrumentation-general-reference_material-list/000009867.md) for additional information on Illumina's Indexed PhiX (1000 Cycle) Control.
2. Use unique index combinations across all lanes.
   1. This ensures that no potential contamination is mistakenly attributed to a sample in an incorrect lane.
3. Confirm the minimum required volume of library is loaded into each sample tube.
   1. **25B** - 275 µL
   2. **10B**, **5B**, **1.5B** - 165 µL

If following the best practices mentioned above, any contamination should be identified or removed during demultiplexing. Otherwise, it may not be possible to accurately identify any potential contamination as indexing is critical for detecting and mitigating cross-lane contamination.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #10118), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000009867%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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