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Unique, dual-matched adapters mitigate index hopping between NGS samples
The document discusses methods to mitigate sample cross-talk in next-generation sequencing (NGS) using unique, dual-matched adapters. It identifies sources of cross-talk such as index hopping and contamination, and highlights the importance of custom dual indices for accurate sample assignment in multiplexed applications. Recommendations for reducing mis-assignment and improving read accuracy in sequencing workflows are also provided.
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Unique, dual-matched adapters mitigate index hopping between NGS samples
- 1. Unique, dual-matched adaptersmitigate index hopping between NGS samples Kristina Giorda, PhD 1
- 2. Outline • NGS workflowand cross-talk • Sources of sample cross-talk and mitigation strategies • Adapter recommendations 2
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- 4. Library construction 4 Fragmentation End repairand A-tailing Adapter ligation Bead cleanup Library amplification Bead cleanup • Y-adapters: 13 bp are complementary • Library conversion of top and bottom strand
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- 6. NGS target captureenrichment 6 • IDT xGen® Lockdown® Probes – Individually synthesized – Individual QC for every probe – Individually normalized – Pooled
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- 8. What is samplecross-talk? 8 Reads are assigned to the wrong sample
- 9. Applications that mightbe impacted by cross-talk • Low-frequency somatic variant detection—false positives from other samples • Ancient DNA research—a single sequence may support DNA survival or contamination • Viral detection—false positives from other samples • Gene expression—bleed over from one sample to another • Microbial profiling—bleed over from one sample to another 9 Kircher M, Sawyer S, et al. (2012) Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic Acids Research, 40(1):e3–e3. D’Amore R, Ijaz UZ, et al. (2016) A comprehensive benchmarking study of protocols and sequencing platforms for 16s rRNA community profiling. BMC Genomics, 17(1):55.
- 10. Custom dual indices 10 AGBT2014 IDT has been making custom dual indices for a long time
- 11. Sources of samplecross-talk • Contamination • Index hopping during multiplex capture • Index hopping during cluster amplification • Misread bases within index sequences • Sample carryover from previous sequencing runs 11 D’Amore R, Ijaz UZ, et al. (2016) A comprehensive benchmarking study of protocols and sequencing platforms for 16s rRNA community profiling. BMC Genomics, 17(1):55.
- 12. Sources of samplecross-talk 12 Index contamination
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- 14. Unique, dual matchedindices reduce contamination mis- assignment 14 • P5 and P7 ligations are independent • Index contamination may occur on any sequencing platform
- 15. Unique, dual-matched adaptersreduce contamination mis- assignment exponentially 15 • 16 libraries were prepared and captured with the IDT xGen® AML Cancer Panel • Libraries were sequenced on the NextSeq® System (Illumina) • 0.09% of reads were filtered out that would have been mis- assigned with combinatorial indices
- 16. Sources of samplecross-talk 16 Index contamination Index hopping Index swapping Index cross-talk Spreading of signal
- 17. Multiplexed target enrichmentindex hopping 17 • Target enrichment index hopping primarily occurs during post-capture PCR • Index hopping may occur on the P5 and P7 side
- 18. Approach for measuringmultiplex capture index hopping • 16 libraries were prepared and 1-, 4-, 8-, or 16-plex captures were performed with the IDT xGen® AML Cancer Panel as using 500 ng per library • Libraries were sequenced on the NextSeq® System (Illumina) 18
- 19. Unique, dual-matched indicesmitigate index hopping during multiplexed target enrichment 19 • There are low levels of index hopping in multiplexed target enrichment • Index hopping reads are filtered out using unique dual-matched indices
- 20. Index hopping readsare effectively filtered out with unique dual-matched indices • Index hopping reads are filtered out with unique dual-matched adapters • Reads would have been mis- assigned with combinatorial indices 20
- 21. Sources of samplecross-talk 21 Index contamination Index hopping Index swapping Index cross-talk Spreading of signal
- 22. Index hopping duringmultiplexed sequencing • Patterned flow cells utilize exclusion amplification (ExAmp) chemistry, associated with more index mis-assignment than bridge amplification 22 Sinha R, Stanley G, et al. (2017) Index switching causes “spreading-of-signal” among multiplexed samples in Illumina HiSeq 4000 DNA sequencing. bioRxiv. www.illumina.com/science/education/minimizing-index-hopping.html
- 23. Index hopping duringmultiplexed sequencing 23 www.illumina.com/content/dam/illumina-marketing/documents/products/whitepapers/index-hopping-white-paper- 770-2017-004.pdf?linkId=36607862 • Index hopping can occur on the P5 and P7 side
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- 25. Sources of samplecross-talk 25 Index contamination Index hopping Index swapping Index cross-talk Spreading of signal Index mis-assignment Demultiplexing noise
- 26. Index design • Errorscan be introduced during synthesis, library preparation, amplification, and sequencing • Need unique sequence tags for each sample • Consider errors (insertions, deletions, and substitutions) • Indices based on edit metric or Levenshtein distance are used to account for substitutions and indels • Need to: avoid homopolymers, match GC content, exclude self-complements, color balance, and consider sequencing platform (4 vs. 2 color) 26 Faircloth BC, Glenn TC. (2012) Not all sequence tags are created equal: Designing and validating sequence identification tags robust to indels. PLOS ONE, 7(8):e42543.
- 27. Edit distance considerssubstitutions and indels 27 Faircloth BC, Glenn TC. (2012) Not all sequence tags are created equal: Designing and validating sequence identification tags robust to indels. PLOS ONE, 7(8):e42543.
- 28. Index quality filtering 28 WrightES, Vetsigian KH. (2016) Quality filtering of Illumina index reads mitigates sample cross-talk. BMC Genomics, 17(1):876. • Quality filtering index reads minimizes cross-talk while preserving the majority of reads • Unique, dual indexing is required for highly sensitive applications • Run-specific and application- specific thresholds can be used to minimize cross-talk
- 29. Unique, dual-matched indicesreduce sample cross-talk 29
- 30. Illumina and IDTpartner on NGS multiplexing and exome enrichment • The proprietary index kits will be compatible with Illumina library prep products and sequencers – Highly optimized for use on platforms with Illumina’s two-channel chemistry and patterned flow cells such as the NovaSeq™ Series – Extend the number of unique dual indexes (UDI) from 8 UDIs to 24 – The new 24 UDI kits can be preordered from Illumina now – Future expansion to 96 UDI kits planned in partnership with Illumina • The optimized index codes are now available through IDT for incorporation into custom adapter orders 30
- 31. xGen® Dual IndexUMI Adapter 31 3-in-1 design
- 32. Conclusions • All sequencingplatforms are susceptible to cross-talk for multiplexed sensitive applications • Unique dual indices mitigate sample cross-talk and enable sensitive applications • IDT has 384 predesigned unique indices for custom adapters – Edit distance ≥3, color balanced as sets of 4, designed for 2- and 4-color sequencers, and with 50% GC content Contact Application Support applicationsupport@idtdna.com for xGen® Dual Index UMI Adapters or help with custom designs 32
- 33. Additional resources • KircherM, Sawyer S, et al. (2012) Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic Acids Research, 40(1):e3–e3. • D’Amore R, Ijaz UZ, et al. (2016) A comprehensive benchmarking study of protocols and sequencing platforms for 16s rRNA community profiling. BMC Genomics, 17(1):55. • Sinha R, Stanley G, et al. (2017) Index switching causes “spreading-of-signal” among multiplexed samples in Illumina HiSeq 4000 DNA sequencing. bioRxiv. • Faircloth BC, Glenn TC. (2012) Not all sequence tags are created equal: Designing and validating sequence identification tags robust to indels. PLOS ONE, 7(8):e42543. • Wright ES, Vetsigian KH. (2016) Quality filtering of Illumina index reads mitigates sample cross- talk. BMC Genomics, 17(1):876. • enseqlopedia.com/2016/12/index-mis-assignment-between-samples-on-hiseq-4000-and-x-ten/ • www.illumina.com/science/education/minimizing-index-hopping.html • www.idtdna.com/pages/support/technical-vault/video-library/next-generation- sequencing/accurate-detection-of-low-frequency-genetic-variants-using-novel-molecular-tagged- sequencing-adapters 33
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