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Best Practices to Minimize Index Hopping

Minimize index hopping in multiplexed runs

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

Increasing Capacity with Multiplexing

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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What is Index Hopping?

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 adapters will see higher levels of index hopping.

Free Adaptor Drives Index Hopping Rates
Effects of Index Misassignment

Effects of Index Misassignment

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

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What is the Impact?

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

Tips and Best Practices

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

  • Remove free adapters 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

Dual Index Sequencing

When pooling libraries for sequencing, we recommend using unique dual indexes over combinatorial dual indexes. Unique dual indexing mitigates index hopping by filtering hopped reads.

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Unique Molecular Identifiers

Unique molecular identifiers are a type of molecular barcoding that provides error correction and increased accuracy during sequencing. Sequencing with UMIs can reduce the rate of false-positive variant calls and increase sensitivity of variant detection.

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Index Hopping Mitigations

Illumina is expanding the number of unique dual indexes we offer, with 24-plex and 96-plex indexes now available. Unique dual indexes enable researchers to remove unexpected combinations and only focus on the ‘true’ data with correct index combinations.

The following products are compatible with the new unique dual index kits:

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

Additional Information

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

Looking for more tips for your next-generation sequencing experiment? View our NGS tutorials for training and best practices.

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Related Solutions

Patterned Flow Cell Technology

Fixed spacing of sequencing clusters with defined feature sizes contributes to increased data output, reduced costs, and faster run times.

Multiplex Sequencing with Index Adapters

Sample multiplexing with index adapters dramatically increases the number of samples analyzed in a single sequencing run.

Illumina University Training

Work with expert Illumina instructors and get hands-on training. We also offer online courses and webinars.

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