The document presents the Xgen® dual index UMI adapters designed for Illumina sequencers, detailing their role in reducing sample cross-talk and improving the accuracy of low-frequency variant detection in NGS workflows. It discusses various sources of sample cross-talk and how unique, dual indexes can mitigate index hopping and contamination issues. The effectiveness of these adapters is highlighted through consensus analysis that enhances variant calling accuracy, significantly lowering false positives and increasing positive predictive values.

1. Dual index adapters with UMIs resolve index hopping
and increase sensitivity of variant detection
Nick Downey, PhD
Applications Scientist
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2. Outline
• Description of new xGen® Dual Index UMI Adapters
• Overview of NGS workflow that includes sample multiplexing
• Discussion of cross-talk
– Sources
– Reduction with dual index adapters
• Discussion of accurate variant detection with UMI error correction
• Additional resources
UMI = unique molecular identifier
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3. New xGen Dual Index UMI Adapter
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3-in-1 design
• Designed for Illumina sequencers
• Compatible with standard, end repair and A-tailing library construction,
including PCR-free library methods
• Reduced sample cross-talk with dual, unique sample indexes
• Ideal for error correction and/or counting applications through use of a
degenerate, 9 bp UMI

4. xGen Dual Index UMI Adapter
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3-in-1 design

5. NGS workflow
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6. Library construction
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Fragmentation
End repair and A-tailing
Adapter ligation
Bead cleanup
Library amplification
Bead cleanup
• Y-adapters: 13 bp are complementary
• Library conversion of top and bottom strand

7. NGS target capture enrichment
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• IDT xGen Lockdown® Probes
– Individually synthesized
– Individual QC for every probe
– Individually normalized
– Pooled

8. What is sample cross-talk?
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• Reads are assigned to the wrong sample

9. Applications that can be 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
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Kircher M, Sawyer S, Meyer M. (2012) Double indexing overcomes inaccuracies in multiplex sequencing on the
Illumina platform. Nucleic Acids Res, 40(1):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, 17:55.

10. Sources of sample cross-talk
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• Index contamination
• Index hopping
• Misread bases
within sample index
• Sample carryover
from previous runs
• Index
mis-assignment
• Index hopping
D’Amore R, Ijaz UZ, et al. (2016) BMC Genomics, 17:55.

11. Combinatorial indexing is susceptible to cross-talk
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12. Unique, dual indexes reduce contamination
mis-assignment
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• Index contamination may occur on any sequencing platform

13. Unique, dual indexes mitigate index hopping
during multiplexed target enrichment
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• There are low levels of index hopping in multiplexed target enrichment
• Index hopping reads are filtered out using unique dual indexes

14. Index hopping during multiplexed sequencing
• Patterned flow cells use
exclusion amplification (ExAmp)
chemistry
• ExAmp is associated with
higher levels of index hopping
than bridge amplification
• PCR-free libraries exhibit higher
levels of index hopping than
amplified libraries
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Sinha R, Stanley G, et al. (2017) Index switching causes “spreading-of-signal” among multiplexed samples in Illumina HiSeq 4000
DNA sequencing. bioRxiv, doi: https://doi.org/10.1101/125724.
Illumina (2017) Minimize index hopping in multiplexed runs. www.illumina.com/science/education/minimizing-index-hopping.html
[accessed November 9, 2017].

15. Unique, dual indexes mitigate cross-talk for
PCR-free libraries
• PCR-free libraries were
sequenced on an Illumina
MiSeq with bridge amplification
• Index hopping or adapter
contamination reads are
correctly filtered out with
unique dual index adapters
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16. Unique, dual indexes reduce sample cross-talk
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17. Co-development of dual index adapters with Illumina
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IDT manufacturers Illumina UDI adapters

18. xGen Dual Index UMI Adapter
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3-in-1 design

19. Circulating cell-free DNA as a “liquid biopsy”
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Bettegowda C, Sausen M, et al. (2014) Detection of circulating tumor DNA in early- and late-stage human malignancies.
Sci Transl Med, 6(224):224ra24.
• Less invasive than
performing a tissue
biopsy
• Theoretically represents
tumor heterogeneity
better than a localized
biopsy sample
• Facilitates on-going,
highly personalized
monitoring

20. Levels of error correction and sensitivity
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21. Consensus analysis
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TP
Total reads
TP
UMI reads

22. Consensus analysis
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TP
Total reads
TP
UMI reads
TP
Consensus reads
(Min3)

23. Consensus analysis
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TP
Total reads
TP
UMI reads
TP
Consensus reads
(Min3)

24. Consensus analysis
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TP
Total reads
TP
UMI reads
TP
Consensus reads
(Min3)

25. Tumor model system
• 25 ng of a 1% mixture (0.5% minimum allelic frequency) was used to
assess sensitivity and positive predictive value (PPV)
• Libraries were captured with a set of custom xGen® Lockdown® Probes
covering 288 common SNP sites for a total target area of ~35kb
• Variant calling was performed with VarDict
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26. Consensus analysis increases variant calling accuracy
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All expected variants
0.2% variant calling threshold

27. Consensus analysis increases variant calling accuracy
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Low frequency variant calling accuracy (≤1%)
54 low frequency variants

28. Oxidative errors are removed using consensus analysis
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Costello M, Pugh TJ, et al. (2013) Discovery and characterization of artifactual mutations in deep coverage targeted
capture sequencing data due to oxidative DNA damage during sample preparation. Nucleic Acids Res, 41(6):e67.

29. Accurate variant detection with UMI error correction
• Without molecular barcoding, it is difficult to distinguish true and
false positives at frequencies below ~1%
• Using UMIs to build consensus reads dramatically increases variant
calling accuracy down to 0.5% using 25 ng of input
– With minimal changes to sensitivity, the number of false positives
dropped ~30-fold using Dual Index UMI adapters
– Considering low frequency variants from 1% to 0.5%, PPV was
increased from 26% to 92%, while keeping sensitivity above 87% using a
variant calling threshold of 0.2%
– Oxidative errors were removed using consensus analysis
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30. Ordering – idtdna.com
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31. Ordering
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32. Index sequences
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33. Index sequences
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Distinct, unique dual indexes

34. Conclusions
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3-in-1 design
• Dual index UMI adapters resolve index hopping and enable accurate
low frequency variant detection
• IDT has 384 predesigned unique indexes for custom adapters
– Edit distance ≥3, color balanced as sets of 4,
designed for 2- and 4-color sequencers and with 50% GC content
– Adapters are made-to-order
• Submit orders to customquotes@idtdna.com
For help with custom adapter designs, contact Application Support
applicationsupport@idtdna.com

35. Additional resources
• Analysis guidelines
• Webinars
• Posters
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36. THANK YOU
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