Precision medicine for oncology requires accurate and sensitive molecular characterization. However, sample degradation, polymerase errors, and sequencing errors reduce accuracy for sequencing genetic variants. By incorporating molecular tagged adapters in target enrichment, and using DNA probes that deliver extremely even and deep coverage, we are able to demonstrate a 300-fold reduction in false positives at or above 0.25% variant frequency. In this presentation, Dr Mirna Jarosz discusses these methods and how they can significantly reduce error rates in your sequencing data.
Accurate detection of low frequency genetic variants using novel, molecular tagged sequencing adapters
1. Accurate detection of low frequency
genetic variants using novel, molecular
tagged sequencing adapters
Mirna Jarosz, PhD
Integrated DNA Technologies, Inc
Webinar—November 16, 2016
2. Outline
• Review
– The growing need for accurate detection of low frequency variants
– Liquid biopsies: what are they, why are they important, and what makes them
challenging?
– Library preparation and target enrichment
• Experimental results
– New adapters containing unique molecular identifiers
– Model system for assessing accuracy of low frequency variant detection
– Analysis methods and accuracy results
2
3. Precision health and oncology
• White House Precision Health Initiative mission statement:
– To enable a new era of medicine through research, technology, and
policies that empower patients, researchers, and providers to work
together toward development of individualized care.
• NCI and cancer.gov define precision medicine as:
– Discovering unique therapies that treat an individual’s cancer based on
the specific abnormalities of their tumor.
From www.cancer.gov
4. Critical-to-know mutation profile to treat lung cancer
Li T, Kung H-J, et al. (2013) Genotyping and genomic profiling of non–small-cell lung
cancer: Implications for current and future therapies. J Clin Oncol, 31(8):1039–1049. 4
5. Sufficient DNA is a challenge, and lung biopsies are invasive
Hagemann IS, Devarakonda S, et al. (2015) Clinical next-generation sequencing in patients with
non–small cell lung cancer. Cancer, 121(4):631–639. 5
6. Outline
• Review
– The growing need for accurate detection of low frequency variants
– Liquid biopsies: what are they, why are they important, and what makes them
challenging?
– Library preparation and target enrichment
• Experimental results
– New adapters containing unique molecular identifiers
– Model system for assessing accuracy of low frequency variant detection
– Analysis methods and accuracy results
6
7. Circulating cell-free DNA as a “liquid biopsy”
7
Bettegowda C, Sausen M, et al. (2014) Detection of circulating tumor DNA in early- and late-stage
human malignancies. Sci Transl Med, 6(224):224ra224.
• Less invasive than performing a
tissue biopsy
• Theoretically represents the full
tumor heterogeneity better than a
localized biopsy sample
• Facilitates on-going, highly
personalized monitoring
8. Demand for higher sensitivity
8
Early detection, monitoring for residual disease, detecting resistance
mutations, tumor profiling when biopsies are not possible
Clin Cancer Res 2014, 20(17):4613–4624
9. Outline
• Review
– The growing need for accurate detection of low frequency variants
– Liquid biopsies: what are they, why are they important, and what makes them
challenging?
– Library preparation and target enrichment
• Experimental results
– New adapters containing unique molecular identifiers
– Model system for assessing accuracy of low frequency variant detection
– Analysis methods and accuracy results
9
14. xGen® Lockdown® Probes are individually synthesized and
QCed
Each xGen® Lockdown® Probe receives an individual ESI-MS analysis
14
Failed Remade
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16. Outline
• Review
– The growing need for accurate detection of low frequency variants
– Liquid biopsies: what are they, why are they important, and what makes them
challenging?
– Library preparation and target enrichment
• Experimental results
– New adapters containing unique molecular identifiers
– Model system for assessing accuracy of low frequency variant detection
– Analysis methods and accuracy results
16
19. Outline
• Review
– The growing need for accurate detection of low frequency variants
– Liquid biopsies: what are they, why are they important, and what makes them
challenging?
– Library preparation and target enrichment
• Experimental results
– New adapters containing unique molecular identifiers
– Model system for assessing accuracy of low frequency variant detection
– Analysis methods and accuracy results
19
22. Outline
• Review
– The growing need for accurate detection of low frequency variants
– Liquid biopsies: what are they, why are they important, and what makes them
challenging?
– Library preparation and target enrichment
• Experimental results
– New adapters containing unique molecular identifiers
– Model system for assessing accuracy of low frequency variant detection
– Analysis methods and accuracy results
22
23. Analysis summary
• 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 performed with VarDict using a threshold variant frequency
of 0.25%
• No UMI analysis uses standard start/stop information to remove apparent
PCR duplicates
• UMI analysis adds back in unique molecules that just happened to share
start/stop sites
• Consensus analysis requires at least three reads from a unique molecule
and uses their consensus as input into variant calling
23
24. Sensitivity and positive predictive value (PPV) with
consensus analysis with 0.25% variant frequency threshold
TP
Total reads
TP
De-dup with start/stop
TP
With UMIs
97
98
99
100
20 40 60 80 100
PPV (%)
Sensitivity(%)
No UMI
(Start/Stop)
UMI
TP
De-dup with UMIs
Mean de-duped coverage
No UMI
(Start/Stop)
0
2000
4000
6000
8000
UMI
24
25. Sensitivity and positive predictive value (PPV) with
consensus analysis with 0.25% variant frequency threshold
TP
Consensus
97
98
99
100
20 40 60 80 100
PPV (%)
Sensitivity(%)
No UMI
(Start/Stop)
UMI
Consensus
Mean de-duped coverage
No UMI
(Start/Stop)
UMI
0
2000
4000
6000
8000
Consensus
25
26. Summary: sensitivity and specificity for SNVs
FP called FP filtered TP called TP filtered TP missing Sensitivity PPV
No UMI
(Start/stop)
641 2 241 0 1 99.59% 27.32%
UMI 368 0 239 0 3 98.76% 39.37%
Consensus only 2 13 239 0 3 98.76% 99.17%
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31. Conclusions
• Without molecular barcoding, it is difficult to distinguish true and false
positives at frequencies below ~5%
• The addition of UMI’s to the ligation adaptors increases unique coverage
due to the rescue of “false” PCR duplicates
• Using UMIs to build consensus reads dramatically increases variant calling
accuracy
– With minimal changes to sensitivity, the number of false positives dropped
~300-fold
– Considering variants down to 0.25% frequency, PPV was increased from
27% to >99%, while keeping sensitivity above 98.5%
31
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33. THANK YOU!
We will email you the webinar recording
and slides next week.