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 -20C. Do not store libraries as a pool.
- Pool libraries just prior to sequencing.