Minimize index hopping in multiplexed runs

Tips and best practices to avoid sequencing read misalignment associated with index switching

Best Practices to Minimize Index Hopping

Improvements in next-generation sequencing (NGS) technology have greatly increased sequencing speed and data output, resulting in the massive sample throughput of current sequencing platforms. A key to utilizing this increased capacity is multiplexing, which adds unique sequences, called indexes, to each DNA fragment during library preparation. This allows large numbers of libraries to be pooled and sequenced simultaneously during a single sequencing run.

Gains in throughput from multiplexing come with an added layer of complexity, as sequencing reads from pooled libraries need to be identified and sorted computationally in a process called demultiplexing before final data analysis. However, with multiplexing, the potential for index hopping is present regardless of the library prep method or sequencing system used. Index hopping may result in assignment of sequencing reads to the wrong index during demultiplexing, leading to misalignment.

Index Hopping Effects and Mitigation Strategies

Gain insights on typical levels of index hopping and the downstream effect on applications.

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Index hopping or index switching is a known phenomenon that has impacted NGS technologies from the time sample multiplexing was developed.1 It causes a specific type of misassignment that results in the incorrect assignment of libraries from the expected index to a different index (in the multiplexed pool).

Index hopping can be seen at slightly elevated levels on instruments using patterned flow cells with exclusion amplification chemistry versus those that do not use patterned flow cells. Libraries with higher levels of free adaptors will see higher levels of index hopping.

Free Adaptor Drives Index Hopping Rates
Effects of Index Misassignment white paper

Effects of Index Misassignment

Learn why it happens and best practices to reduce the impact of index hopping.

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While index hopping can occur, it has a limited effect on most applications and background hopped reads can be filtered out as noise. Typical levels of index hopping on patterned flow cell systems range from 0.1 - 2% depending on the type, quality, and handling of the library. Use of unique dual indexing combinations eliminates hopped reads from downstream analysis because unexpected combinations will be assigned as undetermined.

Index Hopping by Library Type

To minimize the level and effect of index hopping, customers should follow these library preparation best practices:

  • Remove free adaptors from library preps
  • Store libraries individually at -20°C
  • Pool libraries prior to sequencing
  • Use unique dual indexing pooling combinations (unique i5 and i7 indexes)
Index Hopping Rates for 4-plex PCR-Free Library

Illumina is expanding the number of unique dual indexes. The new indexes will launch in a phased approach with 24-plex indexes released first, with a subsequent increase to 96. Unique dual indexes enable researchers to remove unexpected combinations and only focus on the ‘true’ data with correct index combinations.

The following kits will be compatible with the new unique dual index kits:

Illumina is also developing an enzymatic solution that will help reduce the level of free adaptor in libraries. This step takes place after library generation and further reduces index hopping levels.

For more in-depth information, see the following resources:

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References
  1. Kircher M, Sawyer S, Meyer M. Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic Acids Res. 2012:2513–2524.