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

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

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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

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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

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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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