standardized next generation sequencing abundance … dpcr qpcr talk... · 2016-08-01 · cv ngs...
TRANSCRIPT
Standardized Next
Generation
Sequencing
Abundance
Measurements
(StarSeq) using
Competitive
Template Mixtures
AccuGenomics Inc.
dPCR & qPCR 2016
* Nature Biotechnology November 2012
Need for NGS Standardization
dPCR & qPCR 2016
• More Accurate – Controls for library bias
• Improve Throughput – Amplicon convergence
• QA – every target in every sample
Targeted RNA
Sequencing
• QA metrics – sensitivity and precision for every mutation
Targeted DNA
Sequencing
dPCR & qPCR 2016
NORMAL NGS PROCEEDURE
StarSeq Workflow
MULTIPLEX COMPETATIVE
PCR
BARCODE &
ADAPTERS
SEQUENCE
COUNT CT & NT
• Multiplex PCR to saturation converges high &
low abundance targets
• NT:CT ratio maintained throughout library
construction
• Calculate sample abundance from NGS CT
and NT counts
dPCR & qPCR 2016
StarSeq ERCC Reference Standards
• Very good correlation between StarSeq vs. real-time qPCR.
• ROC curves indicate 95% confidence at detecting two-fold or
greater changes.
PLoS ONE (2013) 8(11)
dPCR & qPCR 2016
Multiplex Competitive PCR Converges High
& Low Templates
• PCR conditions converge amplicons toward equimolar concentrations.
• IS:NT ratio maintained while low and high abundance counts
converge.
Abundance
Convergence
PCR
dPCR & qPCR 2016
StarSeq Increases Sample Throughput
TRANSCRIPT ABUNDANCE
REA
DS P
ER
TA
RG
ET
• StarSeq ratio measurements eliminate the existing requirement for abundance
based on total count.
• Convergence allows measurement of targets separated over 8 log concentration
with <1000 counts
PLoS ONE (2013) 8(11)
dPCR & qPCR 2016
Additional Enrichment for Targeted RNAseq
y = 0.9785x - 0.0355
R² = 0.95
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6Flu
idig
m M
ole
cu
le L
OG
ab
un
da
nc
e
Traditional LOG abundance
Transcript Abundance
0
1
2
3
4
5
0 1 2 3 4 5 6
Flu
idig
m S
eq
ue
nc
e L
OG
Co
un
ts/S
am
ple
Sequence LOG Counts/Sample
Sequence Counts Measured
• Addition of Fluidigm Access Array™
• Reduces complications of incompatible primer pairs
• Improves convergence
dPCR & qPCR 2016
FFPE SamplesRNA from FFPE samples – 1 cm2 area x 10 µm (depth) =
1 ug RNA into a 60 uL Reverse Transcription
1 uL of RT into Pre-amp below
(~17 ng of FFPE RNA derived cDNA)
Multiplex Pre-amplification with Target Specific Primers that have Universal Adapter Tails
in the Presence of Competitive Internal Standards
Sta
rSe
q
qPCR
dPCR & qPCR 2016
• More Accurate – Controls for library bias.
• Improve Throughput – Amplicon convergence
• QA – every target in every sample
Targeted RNA
Sequencing
RNAseq Summary
dPCR & qPCR 2016
DNAseq
dPCR & qPCR 2016
Allelic Frequency Testing Effected by
Sampling Issues
Addition of competitive templates ensures accurate target detection
Biomolecular Detection and Quantification (2015) 5:30–37
dPCR & qPCR 2016
Stochastic Error Estimate
EXPECTED
CV
EXPECTED CV
MEA
SU
RED
CV
(n
=4)
Biomolecular Detection and Quantification (2015) 5:30–37
H520 x H23 cell lines mixed at
different ratios and dilutions for 4
SNP measurements (n=4)
dPCR & qPCR 2016
Background Mutations
Introduced by NGS Method
Biomolecular Detection and Quantification (2015) 5:30–37
Sensitive detection of low frequency mutation is confounded by a
variable NGS introduced mutation rate
dPCR & qPCR 2016
NGS INDUCED FREQ DIFFERENCE OF NT - IS
LOG(NT-IS)
Fre
qu
en
cy
-1.0 -0.5 0.0 0.5 1.0
01
00
20
03
00
40
0
Competitive Templates
Competitive template mutation rate estimates NGS background
mutation rate.
Biomolecular Detection and Quantification (2015) 5:30–37
dPCR & qPCR 2016
Targeted DNAseq QA
EXPECTED
CV
NGS
MUTATION
RATE
ALLELIC
FREQUENCY
CONFIDENCE
INTERVAL
ESTIMATE
ALLELIC
LOD
Quality score for every mutation in every sample.
dPCR & qPCR 2016
• More Accurate – Controls for library bias.
• Improve Throughput – Amplicon convergence
• QA – every target in every sample
Targeted RNA
Sequencing
• QA metrics – sensitivity and precision for every mutation
Targeted DNA
Sequencing
StarSeq Benefits
dPCR & qPCR 2016
Acknowledgements
Thomas Blomquist,
M.D./Ph.D.
Erin Crawford, M.S.
Jeff Hammersley, M.D.
Dan Olson, M.D., Ph.D.
Ragheb Assaly, M.D.
Younsook Yoon, M.D.
DA Hernandez
Lauren Stanoszek, B.A.
University of Toledo AccuGenomics, Inc.
Tom Morrison, Ph.D.
Brad Austermiller, B.S.
Nick Lazaridis, Ph.D.
Fluidigm
Luke Stewart