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