The document discusses the importance of accurately detecting low frequency genetic variants through new molecular tagged sequencing adapters and the challenges associated with liquid biopsies. It outlines experimental results showing how unique molecular identifiers (UMIs) enhance the accuracy of variant detection, leading to significantly lower false positives. The conclusions emphasize that utilizing UMIs in sequencing libraries improves overall sensitivity and positive predictive value for variant calling down to 0.25% frequency.

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

10. Sample -> sequencer-ready = library construction
10

11. Library construction
11
Fragmentation
End repair and A-tailing
Adapter ligation
Bead cleanup
Library amplification
Bead cleanup

12. • Detecting low frequency variants requires
ultra-deep coverage
– Whole genome sequencing
– Whole exome sequencing
– Focused Targeted Panels
• IDT xGen® Lockdown® Probes
– Individually synthesized
– Individual QC for every probe
– Individually normalized
– Pooled
NGS target capture enrichment
12

13. Target enrichment using hybridization
13

14. xGen® Lockdown® Probes are individually synthesized and
QCed
Each xGen® Lockdown® Probe receives an individual ESI-MS analysis
14
Failed Remade
Full length
Truncated
Full length

15. Individual synthesis and QC means uniform and complete coverage
15

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

17. Levels of error correction and sensitivity
17

18. Adapter structures
Standard
P5
Dual sample
indexes
P5
A
TA
T
P7
P7
P5
P5
A
TA
T
P7
P7 UMI
UMI
18

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

20. Experimental details
20

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%
26

27. Consensus calling reduces false positives
27

28. Error reduction by base
0
0.02
0.04
A>C A>G A>T C>A C>G C>T
Base substitution
Errorrate(%)
UMI
No UMI (Start/stop)
Consensus
28

29. Deeper consensus data
Family size
Normalizedcounts
Original sample
Deeper consensus coverage
29

30. Deeper consensus drives down C>A / G>T error rate
0
0.01
0.02
0.03
0.04
No UMI
Consensus minimum
Errorrate(%)
Error
C>A
C>T
A>T
A>C
A>G
C>G
2 3 4 5 6 7 8` 9 10
30

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!
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and slides next week.