How to mitigate index hopping

What is index hopping? Index hopping is a specific cause of index misassignment. Index hopping can result in incorrect assignment of libraries from the expected index to a different index in a multiplexed pool, which may lead to misalignment and inaccurate downstream analysis results.

What factors impact index hopping?

• Contamination of free adapters/index primers can lead to increased index hopping.

• The method of library preparation contributes to levels of index hopping. In general, PCR-free library preparation methods that only include adapter ligation generate libraries with higher levels of index hopping than methods that incorporate a subsequent PCR amplification step.

• Libraries clustered on nonpatterned flow cells with traditional bridge amplification typically have lower rates of index hopping (≤ 1%) compared to libraries run on patterned flow cells that use exclusion amplification (ExAmp) cluster generation (≤ 2%).

Best practices to mitigate index hopping:

• Prepare libraries with unique dual index kits, which allow index hopped reads to be bioinformatically removed.

• Remove free adapters from library preps. This may include additional bead clean-up or spin column clean-up if needed.

• Store libraries individually at -20°C. Do not store libraries as a pool.

• Pool libraries just prior to sequencing.

More details about index hopping are found in the white paper Effects of Index Misassignment on Multiplexing and Downstream Analysis.

For any feedback or questions regarding this article (Illumina Knowledge Article #1928), contact Illumina Technical Support [e-mail].