Accurate detection of low frequency genetic variants using novel, molecular tagged sequencing adapters

Mirna Jarosz, PhDIntegrated DNA Technologies, IncWebinar—November 16, 2016

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

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

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

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

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

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):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

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

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

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Sample -> sequencer-ready = library construction

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Library construction

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Fragmentation

EndrepairandA-tailing

Adapterligation

Beadcleanup

Libraryamplification

Beadcleanup

• 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

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Target enrichment using hybridization

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xGen® Lockdown® Probes are individually synthesized and QCed

Each xGen® Lockdown® Probe receives an individual ESI-MS analysis14

Failed Remade

Fulllength

Truncated

Fulllength

Individual synthesis and QC means uniform and complete coverage

15

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

Levels of error correction and sensitivity

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Adapter structures

Standard P5

Dual sample indexes

P5

ATA

T

P7

P7

P5

P5

ATA

T

P7

P7 UMI

UMI

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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

Experimental details

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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

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

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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 100PPV (%)

Sens

itivi

ty (%

)

No UMI(Start/Stop)

UMI

TP

De-dup with UMIs

Mean de-duped coverage

No UMI(Start/Stop)

0

2000

4000

6000

8000

UMI

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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 100PPV (%)

Sens

itivi

ty (%

)

No UMI(Start/Stop)

UMIConsensus

Mean de-duped coverage

No UMI(Start/Stop)

UMI

0

2000

4000

6000

8000

Consensus

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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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Consensus calling reduces false positives

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Error reduction by base

0

0.02

0.04

A>C A>G A>T C>A C>G C>TBase substitution

Erro

r rat

e (%

)

UMI

No UMI (Start/stop)

Consensus

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Deeper consensus data

Family size

Nor

mal

ized

cou

nts

Original sample

Deeper consensus coverage

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Deeper consensus drives down C>A / G>T error rate

0

0.01

0.02

0.03

0.04

No UMIConsensus minimum

Erro

r rat

e (%

)

ErrorC>A

C>T

A>T

A>C

A>G

C>G

2 3 4 5 6 7 8` 9 10

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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%

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