Advances in RainDance Sequence Enrichment Technology and

Transcription

Advances in RainDance Sequence Enrichment Technology and
Advances in RainDance Sequence Enrichment
Technology and Applications in Cancer Research
March 17, 2011
Rendez-Vous Séquençage
Presentation Overview
• Core Technology Review
• Sequence Enrichment Application
• Application to Cancer Research
– Oncology Panel
– UltraDeep/Archival Tissue Sequencing
– Methylation Analysis
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RainDance Technologies
CORE TECHNOLOGY
RainDance Technologies
Who we are:
RainDance Technologies is a Provider of Microdroplet-based Solutions
The Company’s RainStormTM Technology Produces
Picoliter-sized Droplets at a Rate of 10 million Per Hour
Each droplet is the functional
equivalent of an individual test tube
and can contain a single molecule,
cell or reaction
30 micron (16 pL) droplets: 3,000 / second
Accelerating Human Health and Disease Research
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RainDance Technologies
RainDance Droplet Chemistry
RainDance Droplet Characteristics
•
Uniformity
fluorocarbon oil
exterior
•
•
Stability
Control
aqueous
interior
surfactant molecules
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RainDance Technologies
RainStormTM Droplet Technology: MicroFluidic Elements
Generate
Mix
Detect
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Co-Flow
Merge
Timing
Sort
Split
Thermal Zones
Collect
Library
Introduction
RainDance Technologies
SEQUENCE ENRICHMENT
The RainDance Targeted Sequencing Solution
•
Sequence targeted regions of the genome
- Medical Resequencing
- Genome Wide Association Study (GWAS) follow-up
- Candidate genes
- Pathways
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RainDance Technologies
RDT 1000 Reagents:
RDT Assay Inputs
Primer Libraries
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Genomic DNA Template
RDT 1000 Chip
RainDance Technologies
RDT 1000 Reagents: Primer Library
RDT Assay Inputs
Primer Libraries
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Genomic DNA Template
RDT 1000 Chip
RainDance Technologies
Custom Order Primer Libraries
upload gene list
customer
•
•
•
•
RefSeq ID
CCDS
Genomic coordinates
Sequence
• primer design
• oligo synthesis
• primer library production
Primer Library aliquots
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RainDance Technologies
Targeted Resequencing of 200kb Locus
RainDance Coverage: 99.8%
Hybridization Coverage: 63.7%
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RainDance Technologies
Targeted Resequencing of 200kb Locus
RainDance covers regions that hybridization misses
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RainDance Technologies
RainDance Primer Library Formation
1. Primer Design
•
Customer’s regions of interest
Primer Pair
#1
Primer Pair
#2
Primer Pair
#3
Primer Pair
#4
Primer Pair
#5
2. Primer Synthesis
•
Forward and reverse primer pair
3. Primer Droplet Aliquot Manufacture
•
•
Controlled droplet volume
Controlled droplet count
4. Primer Library
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RainDance Technologies
RDT 1000 Reagents: RDT 1000 Chip
RDT Assay Inputs
Primer Libraries
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Genomic DNA Template
Mix
RDT 1000 Chip
RainDance Technologies
Genomic DNA Template Mix
•
Fragmented genomic DNA
– 2 ug of genomic DNA per sample
– Mechanical fragmentation to 2 to 4 Kb size distribution
•
•
•
•
•
PCR reaction components except the primers
–
–
–
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Nebulization
Hydroshear
Covaris
Bioruptor
DNA polymerase
dNTP’s
buffer
RainDance Technologies
RDT 1000 Reagents: RDT 1000 Chip
RDT Assay Inputs
Primer Libraries
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Genomic DNA Template
Mix
RDT 1000 Chip
RainDance Technologies
RDT Polymerase Chain Reaction
Output
Inputs
Genomic DNA Template Droplet
33 microns - 18 pL
5’
3’
fwd
rev
3’
5’
Primer Pair Droplet
25 microns - 8 pL
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Merge
PCR Droplet
37 microns - 26 pL
RainDance Technologies
RDT 1000 Droplet-Based PCR
Primer
Library
Genomic DNA
Template Mix
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RainDance Technologies
RDT 1000 Droplet-Based PCR
Primer
Library
Genomic DNA
Template Mix
2 million droplets
45 minutes per run
Droplet
Merge Area
PCR Library
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RainDance Technologies
APPLICATIONS IN CANCER
BIOLOGY
Solutions for Cancer Research
• Cancer Pathways Gene Network
• DeepSeq™ FFPE Solution
– Rare Mutation Detection (1%)
– FFPE or Fresh Frozen
– Very easy workflow
• MethylSeq™ Solution
– Direct quantitation from bisulfite-treated DNA
– Allele-specific, single-base differences
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RainDance Technologies
Cancer Pathways Gene Network
Screen cancer pathway genes and
networks with high fidelity and efficiency
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Company Confidential
RainDance Technologies
Cancer Pathways Gene Network Panel
• 4000 amplicon library
• 142 Oncogenes
– Coding exons
– 5’ and 3’ splice sites
– 3’UTR
Performance
Sample A
Sample B
– Promoter
(1kb)
Average Reads per Base
1,074
308
EGFR
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Specificity
75.5%
75.4%
20X Coverage
98%
97%
SNP Concordance
(Homozygous)
99.7%
99.7%
SNP Concordance
(Heterozygous)
99.3%
98.9%
RainDance Technologies
DeepSeq™ FFPE Solution
Discover more rare cancer mutations
using previously inaccessible samples
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RainDance Technologies
DeepSeq™ Solution
• 500 amplicons (50-100 genes)
– Interrogate any region of the genome: exons, non-coding, repeats, high
G-C, splice junctions, regulatory regions, and high homology
• Up to 50,000x coverage per loci with C20 sequencing
• FFPE or fresh frozen – unlock vast sample collections
• Easy workflow: 2 days  6 hours
– Tailed primers eliminate library prep, concatenate and shearing
– Barcodes PCR-ed in for up to 12 samples per sequencer lane
• Cost ~$500 including barcoding next-gen sequencing
Discover novel mutations prevalent at 1%
in heterogeneous tumor samples
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RainDance Technologies
RainDance 2-Step Indexed Tailed Primer Workflow
Tailed
Primer Library
RDT 1000 Instrument
Step 1: Tailed Primers
Step 2: Universal PCR
Step 1
B
Step 2
A
C
Gene Target
B
Gene Target
A’
Target Specific Tailed Primers
A – Target Specific Sequence
B - NGS Adaptor Sequence
B’
B’
C’
Universal Primers
B - NGS Adaptor Sequence
C - NGS Adaptor Sequence + barcode
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RainDance Technologies
Significantly Simpler Workflow: 6 Hours
RainDance Today
DeepSeq FFPE
Genomic DNA
Shearing
20 minutes
Genomic DNA
Shearing
RDT 1000 Merge
45 minutes
RDT 1000 Merge
PCR
180 minutes
Concatenation &
Shearing
1 day
Illumina Library
Construction
150 minutes
PCR
Universal PCR
less than 6 hrs
30 minutes
Savings of
over $350
2 days
Sequencing
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Sequencing
RainDance Technologies
Ultra-Deep 1% Rare Mutation Detection
500 amplicon library
Test of Limits of Detection: Blend of DNA from HapMap samples
Chr
Ref
SNP
Expected
%
Obs%
# Total
Reads
#A
#C
#G
#T
Chr11
C
T
1%
1.2%
35,456
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34995
24
413
• Average reads per base: 32,000x
• 70% of SNPs between 0.5x and 2x of mean
• 98% of mapped reads on target
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RainDance Technologies
High Performance with FFPE and WGA FFPE
Calculated
Trace
High quality Promega DNA
• 2x100bp paired end on Illumina HiSeq
• Constrained amplicon size to 200 bp
• High Specificity (~98%)
3 ug Liver FFPE DNA
2 ug WGA DNA from
100 ng starting FFPE DNA
1st
Step
2nd
Step
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RainDance Technologies
DeepSeq FFPE Solution Performance
% of Target Bases Covered at
Samples
Total
Reads
Mappable
Specificity Mean Base
Reads
(%)
Coverage
(%)
Breast
9,369,774
(FF-NAT)
99.7%
Breast
(FF)
6,943,080
99.7%
Breast
(FFPE)
9,267,436
94.2%
14,219
Sample94.4%
10,459
Breast93.3%
94.7%
13,365
C20
C100
C1000
C2000 C10000
99.6%
99.4%
99.1%
98.5%
98.2%
77.6%
97.8%
Concordance
99.1%
98.2% 97.3%
50.0%
96.9%
62.0%
96.6%
99.4%
SNPs99.6%
detected
Colon
Coverage
Uniformity*
C1
45/45
99.6%
99.6%
(FF/FFPE)
99.2%
50/50
100% 98.2%
98.7%
100%
Colon
(FF)
12,364,728
99.8%
95.2%
18,851
99.6%
99.2%
99.1%
97.6%
94.9%
66.0%
87.5%
Colon
(FFPE)
12,715,013
98.7%
93.2%
19,014
99.6%
99.6%
99.2%
99.1%
98.7%
89.3%
97.2%
*% of bases > 0.2x of the mean
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RainDance Technologies
Low Frequency Mutations found in FF and FFPE
COSMIC Cancer Genome Browser
Cancer Genome Project, Welcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK Tel +44 (0)1223 834244
Sample
Genomic
Coordinate
(GRCh37/hg19)
Frequency of Mutation
COSMIC ID
(v51)
FF-NAT
FF
FFPE
Breast* chr3:178,952,085
not
found
25.2%
32.6%
776
Colon
N.A.
16.1%
29.7%
13127
chr5:112,175,639
Gene
Nucleotide
Mutation
Protein
Change
Deleterious
Mutation
PIK3CA
c.3140A>T
p.H1047L
(missense)
(Unknown)
APC
c.4348C>T
p.R1450*
(nonsense)
Deleterious
(Known)
*This SNP was not detected in the normal adjacent, fresh frozen B2 sample. No adjacent normal sample was available for C2.
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RainDance Technologies
MethylSeq™ Solution
Discover epigenetic variations associated
with cancer and other complex diseases
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RainDance Technologies
MethylSeq™ Advantages
•
Discover more epigenetic variations
•
Leverage sophisticated primer design algorithm
•
Measure multiple cytosine residues in a single CpG island
on the same DNA strand
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•
Eliminate competitive amplification bias
•
Achieve significant time and cost savings
•
Achieve results and publish faster than ever before
RainDance Technologies
Targeted Methylation of CpG Islands
% Methylation of Promoter Regions
Quantitative analysis of methylation with single base resolution
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RainDance Technologies
Targeted Sequencing of CpG Islands for 50 gene study
Methylation detection >100 reads at each CpG site
Wild type
MTas
treated
2902/2967 CpGs covered (97.8%)
2944/2967 CpGs covered (99.2%)
% Methylation
~20% wild-type fully methylated
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MTas treated fully methylated
RainDance Technologies
THANK YOU
Jeff Fitzgerald
fitzgeraldj@raindancetech.com
(413) 245-9248