Integrated Solutions for Automated Next Generation Sequencing

Integrated Solutions for
Automated Next Generation
Sequencing
From Sample to Sequence
Young K. Yun, Ph.D.,
PekinElmer Inc.,
Sr. Director, International Business
development
1
© 2009 PerkinElmer
Who is Caliper Life Sciences?
Summer 2003
26 years of Laboratory Automation
Inovation for the Life Sciences Industry
11 years of leadership in Microfluidics
and Lab-on-the –Chip technologies
2
3
Caliper Life Sciences: Worldwide
Offices
(Sales, Marketing and Technical Support)
Hopkinton, MA
(Global Headquarters)
Mountain View,CA
Alliance, Nebraksa
McCook, Nebraska
*United Kingdom
*France
*Benelux
*Germany
*Switzerland
*Japan
Cranbury, NJ
El Paso, TX
Hanover, MD
Alameda, CA
450 employees
4
PerkinElmer & Caliper …A Powerful Combination…
Strong global sales channels,
profitability and cash flow
Leading market positions –
newborn, analytical, service &
drug discovery
Strong informatics, one source
and reagents
Gaps in molecular platforms, next
gen sequencing, in vivo imaging
Market Leader in Drug
Discovery & Diagnostics
Comprehensive Product
& Services Suite
Innovative & Customer-Centric
Culture
Worldwide Sales Presence
Strategically Positioned in High
Growth Markets
Strong vision / momentum in
personalized medicine
Leader in molecular imaging,
microfluidics, in vivo imaging
and assays
Strong Innovation, growth &
customer centric culture
Limited presence in emerging
markets and Dx infrastructure
Strengthened PerkinElmer with advanced technologies and
capabilities to serve World Health end markets
…with the potential to revolutionize medicine
5
PerkinElmer & Caliper …A Powerful Combination…
“We are very excited about the tremendous
opportunity we have to revolutionize and
personalize medicine and world
health! Working together, we will be able to
have a powerful, transformative impact on
the world”
Kevin Hrusovsky, President of Life Sciences and
Technology, PerkinElmer
6
7
Our Platforms for Translational Research
Clinical
Instruments
Maestro, Vectra Systems
Reagents
& Services
Human
NGS
Fluorescent agents
SeeGene
Tissue
Operetta (HCS) systems
EnSpire multimode detection
Automation, cell plate
LabChip technology
Zephyr & JANUS
Automation
EnVision plate readers
Labels & Markers,
TSA reagents,
Tissue Services
Small Animal
Cell
Preclinical
DATA QUALITY
FMT & IVIS systems
Labels & Markers,
Fluorescence, Bioluminescence
GPCR’s
Labeled Cells & Markers
Rads, Alpha, LANCE, DELFIA Assays
DNA, RNA,
Protein
AlphaLISA, Rads, LANCE, DELFIA reagents
In vitro
In vivo
COST OF TESTING
8
Building The In Vitro To In Vivo Bridge
In vitro
• LabChip technology
• CRi
FMT imaging systems
& agents
IVIS imaging systems
Pre-clinical
Operetta HCS systems
AlphaLISA assays
Radiometric detection
TSA reagents
Pre -clinical
Pre -clinical
•
•
•
•
In vivo
Pathology
Digital Pathology
Clinical and companion diagnostics
Molecular and Imaging technology
Enabling personalized medicine
Clinical
In vitro
Clinical
Clinical
• Prenatal and neonatal screening
• Molecular Dx – DXI/DXII
• Circulating tumor cells
In vivo
9
Life Sciences & Technology
LIFE SCIENCES & TECHNOLOGY
INFORMATICS
Services, Reagents &
Informatics
Discovery & QC Assays
GENOMIC ANALYSIS
BIOTHERAPEUTICS
TARGETED SMALL
MOLECULE
IMAGING & PATHOLOGY
Detection & Probes
Readers & Reagents
BIOMARKERS
CELLULAR SYSTEMS
Diagnostics
INFORMATICS
HCS Imaging
& consumables
10
Targeted Small Molecules: Helping you find leads
EnSpire ®
Multimode Plate Reader with
Epic Label-Free Technology
Multilabel
High Content
Label-free
Opera™
High Content Screening
JANUS® Automated Workstations
with EnVision® Multilabel Plate Reader
cell::explorer
plate::explorer
Automation
Solutions
Detection
Automation
AlphaLISA
LANCE Ultra
AequoScreen
HTS
LEADS
DRUGS
Assays
AlphaLISA® Assays
LANCE® Ultra Kinase Assays
AequoScreen® Calcium Flux Assays
… focused on differentiated targets (e.g.11
Biotherapeutics are Transforming Treatment Regimens
Faster
Discovery
Higher Accuracy
To Patient
JANUS automated
workstations
with EnVision multilabel plate
reader
EnSpire multimode
plate reader with
label-free technology
AlphaLISA Assays
DELFIA Assays
BriteLITE
AlphaLISA
Assays
Target
Identification
Target
Validation
Clonal
Screening
Safety
Testing
Clinical
Testing
Phase 1
Trials
Phase 2
Trials
Phase 3
Trials
Functional
Testing
Protein
Purification &
Manufacture
Formulation
Engineering Development
& QC
LabChip GXI
nucleic acid separations
system
Manufacturing
Sciclone G3
automated workstation
Shorter Development Time
LabChip GXII
protein analysis and
quantitation system
Affordable Technologies
12
High Content, Cellular Systems Provide Relevant Disease Data
3D Analysis in
Neuroscience Research
High Content Imaging in Stem Cell Research
Automated High Content Screening in Cancer Research
… bridging in vitro to in vivo
PerkinElmer Technology was Instrumental in these Discoveries
FDA Approved
Pfizer, Sutent (sunitinib)
(kidney and stomach cancer)
Pfizer, Lyrica (pregabalin)
(HIV)
Pfizer, Celsentri (maraviroc)
(neuropathic pain)
Genentech, Zelboraf (vemurafenib)
(melanoma, BRAF V600E mutation)
Novartis, Tasigna (nilotinib)
(CML, chronic myeloid leukemia, Gleveec resistance)
Novartis, Zometa (zoledronic acid)
In Clinical Trials
Phase I:
Cell Genesys, CG0070 (Bladder and multiple indications)
Nereus Pharma, NPI-0052 (multiple myeloma)
Cephalon, CEP-26401 (Alzheimer’s & schizophrenia)
Millennium, PS-341 (combo) (Non-Hodgkin’s Lymphoma, others)
Insert Therapeutics, IT101 (solid tumors)
Novartis, CHIR-258 (Metastatic melanoma)
Novartis, AEE788 (Advanced Cancers)
(metastasis of breast, lung, prostate and multiple myeloma)
Takeda, Rozerem (ramelteon)
(sleep disorders)
BMS, Sprycel (dasatinib)
Phase II,III:
Sanofi-Aventis, Aflibercept (Multiple indications)
Boehringer Ingelheim, Oldodaterol (COPD)
(CML, chronic myeloid leukemia, Gleveec resistance)
SIRTex, SIR-Spheres (yttrium-90)
(liver cancer)
Cubist Pharma, Cubicin (daptomycin)
Xencor, XmAb2513 (Hodgkin’s Lymphoma)
Neurogen, DAB-452 (Parkinson)
EntreMed, Panzem
(Recurrent Glioblastoma)
(S. aureus infections – MRSA treatment)
14
Traditional Pathology is “Prone to Error”
Traditional H&E
Single breast cancer marker analysis is “prone to error”
90,000 Ductal Carcinoma in situ (D.C.I.S.) cases
misdiagnosed
Her2 IHC status alone: 22% false positives and 9% false
negatives
ER / PR status alone – 10-20% false positive
* New York Times, Prone to Error: Earliest Steps to Find Cancer, July 19th, 2010
Multiplex Biomarker analysis
Multiple biomarker classification for breast cancer prognosis
/ treatment
Quantitative, structural, multiplex biomarker data can
improve diagnosis
Reduced false positives and false negatives
… improving clinical15
Biomarkers are a Key to Translational Sciences
Can only tell that a
particular antigen is
present, not that the
antibody was bound
to that antigen.
Shows location of
fluorophore inside
tumor.
This is critical for
validation in vivo results
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Agent Portfolio: Rapidly Expanding Across Key Biomarkers
Oncology
Cardiovascular
Hypoxi
Folate
a
Her2/N
eu
Annexi
n
Integrin αvβ3
Liver Toxicology
Adaptation
of select
serum
Infectious disease
Bacteri
a
Arthritis
Hypertension
Cathepsins B,
L, S
MMPs 2, 7, 9,
Vascula
13
r
Neutrophil
Elastase
Cathepsin K
Hydroxyapatit
e
Gastric Emptying
markers for
in situ
imaging
Inflammation
Bone Biology
Physiologic
Reni
n
… PerkinElmer partners to rapidly develop new in17
Collaborating with MGH on Biomarkers for CTCs
CTC
Primary Tumor
“The possibilities and promise of CTC research are very
exciting...they may pave the way to a reliable surrogate
biomarker for cancer, leading to new early detection
tests for quicker intervention – and better patient
outcomes and prognosis.”
Metastasis
Finding 1 cancer cell among 1 billion healthy
cells
4X
VectraTM
60X
CTCs
CTC Analysis with MGH Herringbone-Chip and Perkin Elmer Vectra
… oncologists detect, monitor and treat cancers in the future
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NGS: Debottleneck Sample Prep & Stratify Patients
Geospiza LIMS Sample Tracking
Isolate
Sample
MBW
DS
JANUS & chemagen
Generate
Library
Fragment
& Sizing
XT
GX
NGS
Quantify
Sample
XT
GX
Sequence
NGSW
Analyze
Geospiza
Zephyr Molecular Biology Workstation (MBW)
JANUS Solutions and chemagen • Nucleic Acid Extraction
LabChip DS System • UV-Vis DNA/RNA Quantification
LabChip XT System • Automated DNA Fractionation and Sizing
LabChip GX System • HT Bioanalyzer-like QC for DNA and RNA
Sequencing Service
Sciclone NGS Workstation • Automated Highthroughput Sequencing Sample Preparation
Smaller samples, better quality & reduced PCR bias,
higher throughput & productivity with lower costs,
informatics correlation linkages
Zephyr NGS Workstation
(NGSW) • Post PCR Workstation
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Tumor Heterogeneity and Personalized Medicine
Challenges
Hard to characterize – disease profile
varies across body
Hard to treat – multiple molecular targets
PerkinElmer Solutions
Only 1/3 of all mutations were present in all 14 kidney tumor
sites – there were more differences than similarities within the
SAME tumor
A prognostic gene signature in one region of a tumor had a
GOOD prognosis, whereas another region of the same tumor
had a BAD prognosis!
Heterogeneity helps explain why many treatments eventually
stop working
Assess multiple sites simultaneously via
CTCs, tumor FNAs and lymph node
Molecular tests compatible with FNAs,
multiple biopsies less invasive
More information from each sample multiplexing with MSI
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NGS Identifies Tumors that may Respond to Sunitinib, Sutent
2% of lung tumors may respond
to Pfizer’s cancer drug Sutent.
53% colon and 71% lung tumors
have mutations that may
respond to currently available
cancer treatments
21
AVATAR Scan System Approval Plan
Institute for Refractory Cancer Research (IRCR)
SAMSUNG MEDICAL CENTER
Institute for Refractory Cancer Research
22
AVATAR Scan system: A Solution for personalized cancer therapy
Avatar Cell Library
AVATAR Scan System
Patient derived cell
Automatic HTS
Genome DB
Clinical DB
Ideal Combination
Anti-cancer Drug Library
Chemical and Functional Genomics
FDA Approved Drug Library
In vivo Functional Validation
Personalized Therapy
Chemical Library
“AVATAR”
Overcome Drug
resistance
Drug Response Algorithm
shRNA or ORF Library
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AVATAR Scan System 구성
1.환자유래 암(줄기) 세포 분리 시스템
2.환자 유래 암 (줄기) 세포 배양 시스템
3.환자 맞춤형 항암제 스크리닝 시스템을
포함하는
High Throughput Screening(HTS) 시스템
4.동물 모델를 이용한 환자 맞춤형 항암제 검증
시스템
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AVATAR Scan System(Personalized Drug Screening System)
Avatar Cell Library
AVATAR Scan System
Patient derived cell
Automatic HTS
Genome DB
Clinical DB
Ideal Combination
Anti-cancer Drug Library
Chemical and Functional Genomics
FDA Approved Drug Library
In vivo Functional Validation
Personalized Therapy
Chemical Library
“AVATAR”
Overcome Drug
resistance
Drug Response Algorithm
shRNA or ORF Library
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AVATAR Scan System(Personalized Drug Screening System)
Personalized Drug Screening System 정의
• 본 기술은 환자의 체내 환경을 상당히 정확하게 모사
할수
있어, 환자에게 최적의 효능을 나타내는 환자
맞춤형 항암제를 효과적이면서도 경제적으로 선별 할
수 있을것이므로 맞춤형 의료 서비스 분야을 선도 할
수 있는 기술임
26
AVATA Scan System(Personalized Drug Screening System)
목표
•환 자 맞 춤 형 항 암 제 스 크 리 닝 기 술 공 정 을 의 료
목적으로
활용 할 수 있도록 식약처에 의료 기기 승인 요청
필요성
•개인 맞춤형 항암치료에 관한 unmet medical needs
•개인 맞춤형 항암치료제 스크리닝 인터페이스 기술의
unmet needs
•이를 위해 검증된 시스템 필요성 대두
27
AVATAR Scan System의 승인을 위한 전략적 정의
제조사에서 생산된 자동화 시스템
Work Flow
2
4
제조사: PerkinElmer
1
3
3
1
3
4
3
2
제조사: Liconic
Instrument
제조사: PerkinElmer
4
제조사: Thermo Fisher
1.
2.
3.
4.
Janus: Automated Liquid Handing System
EnVision: Multilabel Reader
Orbitor RS Microplate Mover
Microtiter Plate Incubator
Action Item
1.PerkinElemer meeting 추진 하여 각각의 장비에 대한 의료 기기 승인 요청
28
29
Today: LIMS & Analysis for NGS
Informatics
RNA-Seq
ChIP-Seq
Me-Seq
Reseque
ncing
30
New Pathology Workflows for Next Gen Sequencing
Clinical NGS needs sample prep methods that collect tumor tissue from
biopsies, and measure % malignancy of collected tissue
Knowing % malignancy enables reliable and economical NGS
Combining digital pathology, automation and robotics
Validation study in process University of Pennsylvania
Scan Slides
Panoramic 150
Analyze
%
malignancy
Robotics collect
tumor from FFPE
block
Tumor in tube
or plate for
NGS
TMA Grand Master
31
Geospiza Future Direction
Raw Data
Variant Analysis Medical Treatment Plan
Curated Literature
32
Drug Combination Program Overview
Drug combinations are relevant
 Address multi-factorial nature of disease
 Overcomes redundant signaling pathways
 Counteracts biological buffering &
compensation mechanisms
Drug combinations may provide
advantages
 Increased benefit; larger therapeutic window
 Lower toxicity and side effects
 Minimized risk of developing drug resistance
CDAS Drug Combination Expertise
 Experimental study design (rational/random)
 High throughput data analysis and
Interpretation
 Custom vitro/vivo follow up
www.cellsignal.com
33
Small animal Imaging: Non-invasive Functional/Anatomical Imaging
In Vivo microCT
High ThroughputHigh Throughput
Bioluminescence Fluorescence
Spectral
Unmixing
Caliper
Automatic
Tomographic
Co-registration
Cerenkov
Imaging of
Radioisotopes
Fluorescence
Tomography
PerkinElmer
Tissue imaging
Cellular imaging
…a spectrum of opportunities
34
Advances in Sequencing Technology
Sequencing Cost prohibitive
Alternative Detection Schemes (microarrays)
Lower Sensitivity
Hard to Scale: Single Sample/Detection
Sequencing Cost Effective:
Alternative Sample Preparation Schemes
Higher Sensitivity
Scalable: Sample Multiplexing
35
Enabling Science
NGS: Swiss Army Knife for Life Science Research
•
•
•
•
•
•
•
•
•
Exome Sequencing
Diagnostic panels
ChIP-Seq
Bisulfite Sequencing
RNA-seq
smallRNA & miRNA seq
De Novo Assembly
Viral Sequencing
WGS
• Mutation Discovery
• Structural Variants
• GWAS
36
NGS Application Expansion
37
Leveraging High Throughput
Systems and Applications
Application
PGM
MiSeq
HiSeq
SOLiD
√
√
√
√
454
PacBio
WGS
Comparative (Euk)
Microbe (Pro./viral)
√
√
√
Structural (Euk)
Whole Exome
Targeted Seq
√
√
√
RNA-Seq
Small/microRNA
√
√
ChIP-Seq
√
√
1Gb
2Gb
Throughput
√
√
√
600Gb
200Gb
√
√
√
√
√
√
√
√
700 Mb
Bench-top Sequencer Compatible Applications
Amplicons
100 - 2000 b
38
Plasmids
Viruses
5 - 10 kb
Bacteria
Yeast
2 - 6 Mb
12 Mb
Human
Human
Exome
Genome
50 Mb
3 Gb
Bottleneck Sample Preparation
Source: The Global Outlook for Next Generation Sequencing: Usage, Platform Drivers & Workflow (2011) BioInformatics LLC. Survey
asking 267 scientists currently using Next Generation Sequencing.
39
NGS Sample Prep: Junk In, Junk Out
40
Sample Prep Tied to
Focus on High Quality Applications
41
Standardization of Core Processes
mRNASeq
ChIPSeq
Isolate RNA
Fix + Lyse cells
Enrich mRNA
Fragment mRNA
Prepare cDNA
Fragment
DNA:Protein
Capture w/antiHistone Ab
Elute + Isolate
DNA
MeDIPSeq
DNASeq
Sequence
Capture
Methyl
Seq
Isolate DNA
Isolate DNA
Isolate DNA
Isolate DNA
Fragment DNA
Fragment DNA
Fragment DNA
Bisulfite
Treatment
Capture w/MBP
Size-Select DNA
Elute
End Repair
All applications utilize the
core library prep process at
some stage in the workflow
A-Tail
Normalize
Adapter Ligation
w/barcodes
Adapter Ligation
PCR
PCR
Capture w/Probes
Elute
PCR
Pool samples
Pool samples
NGS
42
43
General NGS Workflow
Caliper Technologies in NGS Workflow
Automation
Sciclone NGS WS and Zephyr NGS WS
Isolate
Sample
Isolation/Purification
DNA and/or RNA
*
Shear
Sample
Generate
Library
Shear/Fragment
Small fragments
Library Generation
- Enzymatic reaction
- Purification
*
Automated Sample Preparation
Quant.
Sample
Sequence
Quantization &
Sample Analysis
Purity and integrity
Sequencing
Seq. chemistry
Microfluidics Analysis
Sequence
Capture
LabChip GX
High Throughput Analysis of DNA and RNA
Microfluidics Preparative
LabChip XT
Automated Nucleic Acid Fractionation and Sizing
44
mRNASeq
ChIPSeq
Isolate RNA
Fix + Lyse cells
Enrich mRNA
Fragment mRNA
Prepare cDNA
Fragment
DNA:Protein
Capture w/antiHistone Ab
Elute + Isolate
DNA
MeDIPSeq
DNASeq
Sequence
Capture
Methyl
Seq
Isolate DNA
Isolate DNA
Isolate DNA
Isolate DNA
Fragment DNA
Fragment DNA
Fragment DNA
Bisulfite
Treatment
Capture w/MBP
Size-Select DNA
Elute
End Repair
A-Tail
All applications utilize the
core library prep process at
some stage in the workflow
Normalize
Adapter Ligation
w/barcodes
Adapter Ligation
PCR
PCR
Capture w/Probes
Elute
PCR
Pool samples
Pool samples
NGS
45
General NGS Workflow
Caliper Technologies in NGS Workflow
Automation
Sciclone NGS WS and Zephyr NGS WS
Isolate
Sample
Isolation/Purification
DNA and/or RNA
*
Shear
Sample
Generate
Library
Shear/Fragment
Small fragments
Library Generation
- Enzymatic reaction
- Purification
*
Automated Sample Preparation
Quant.
Sample
Sequence
Quantization &
Sample Analysis
Purity and integrity
Sequencing
Seq. chemistry
Microfluidics Analysis
Sequence
Capture
LabChip GX
High Throughput Analysis of DNA and RNA
Microfluidics Preparative
LabChip XT
Automated Nucleic Acid Fractionation and Sizing
46
Sciclone NGS & Zephyr NGS
Automated Sample Preparation
47
Zephyr/Sciclone NGS Sample Prep & Library Engine
Sequence Capture
-
Library Generation
-
RNA preparation
-
SPRI Bead Purification
+
Sciclone NGS
Zephyr NGS
48
Pre and Post PCR
Liquid Handling
Sheared DNA
Size Selection
Pre PCR
Post PCR
Library Generation
Clean-Up
LHI: Sciclone
Amplification!
Clean-Up
Ready for Sequencing
LHII: Zephyr
49
Sequencing: Automated Library Preparation
Pre PCR
Sciclone NGS Workstation
Complete library production
Capabilities
• 96 libraries in 4 hours;
• Flexible chemistry and methods
• Higher DNA yields
• Scalable solution; walk away time
• Device Integration :
• Twister II for capacity
• LabChip GX for QC
• Thermocycler
• Covaris shearing
• qPCR
Methods
• RNA preparation: Illumina & Nugen
• Library construction: SOLiD, Illumina, NEB, Nextera
• Target enrichment: SureSelect & SeqCap EZ
Post PCR
Zephyr NGS Workstation
NGS Post PCR Sample Preparation
Capabilities
• SPRI purification protocol
• Normalization protocol
• Pooling Protocol
• Pre-programmed methods
50
Sciclone G3 NGS Workstation: Layout
Heated Shaker
Magnet
Reagent Storage
1. Random Primers
2. Polymerase, etc.
Orbital Shaker
24 Deck Positions
Different NGS Applications, Different NGS Systems…but Same Hardware Deck Layout!
51
Preliminary Specs
Configuration
•
•
•
•
•
96 High Volume Head
Left gripper
1 x 4-110°C temperature controlled locator
1 x 4-70°C shaking locator
Removable Magnet locator
Applications
Initial methods will address post PCR steps
including:
• Post PCR SPRI cleanup
• QC plate creation
- LabChip GX
- 96 & 384 qPCR setup
• Normalization
• Pooling of Indexed Libraries
Future applications will support amplicon
sample prep for small NGS devices
(MiSeq, PGM, GS Jr)
52
Flexible Tip Loading and Tip Management
Capability to load tips as needed based on number of samples
53
Single Tip Loading Using 96 Channel Head
Single tip loading for master mix broadcasting and
normalization routines
54
Sciclone NGS Control Software
Full-Featured Method Editor
Simplified GUI Interface
Methods are easily modified for customized workflows
55
Sciclone NGS Control Software
Modular method design is easily modified for customized workflows
56
Sciclone NGS Control Software: Runtime Interface
Simple and intuitive interface
57
Sciclone NGS Control Software: Setup GUI
58
Sciclone NGS Control Software: Setup GUI
59
Sciclone NGS Control Software: Setup GUI
60
Sciclone NGS Control Software: Setup GUI
61
Sciclone NGS Control Software: Setup GUI
62
Automated mRNA Seq Library Prep on Sciclone NGS
(Illumina TruSeq)
Purified TruSeq Libraries
Total RNA
8-96 samples per run
< 30 min hands-on time
1000
800
600
400
Average = 601
SD = 67
200
96
84
72
60
48
36
24
12
0
0
Day 1:
- mRNA purification
- Fragmentation
-1st and 2nd strand synthesis
Day 2:
- End repair
- A-tailing
- Ligation
-PCR enrichment
-Normalization and pooling
mRNA Seq Library Yield
Yield (ng)
•
•
Sample Number
63
Sciclone NGS
Zephyr
64
Sciclone NGS Workstation Application Roadmap
Category
Vendor
Application
Status
Fragment Library Prep
Illumina
TruSeqTM DNA Sample Prep
Complete
Fragment Library Prep
NEB
NEBNext® DNA Sample Prep Master Mix Set 1 (Illumina)
Complete
Sequence Capture
Agilent
SureSelectXT for Illumina Paired-End Sequencing
Complete
Fragment Library Prep
Life Technologies
SOLiDTM Fragment Library Prep
Complete
Sequence Capture
Nimblegen
SeqCap EZ Exome SR for TruSeq Libraries
Complete
mRNA Seq
Illumina
TruSeq RNA Sample Prep
Complete
Fragment Library Prep
NuGEN
Encore NGS Library System I (Illumina)
Complete
mRNA Seq
NuGEN
Ovation RNA-Seq System
Complete
Fragment Library Prep
NEB
NEBNext DNA Sample Prep Master Mix Set 3 (SOLiD)
In Validation
Sequence Capture
Agilent
SureSelectXT for SOLiD Sequencing
In Validation
Fragment Library Prep
454
Rapid Library Prep
Ready for Validation
mRNA Seq
Life Technologies
SOLiD Total RNA – Whole Transcriptome
Ready for Validation
Small RNA Seq
Illumina
TruSeq Small RNA Sample Prep
Ready for Validation
Fragment Library Prep
Epicentre
Nextera™ DNA Sample Prep Kit
Ready for Validation
Fragment Library Prep
NEB
NEBNext DNA Sample Prep Master Mix Set 2 (454)
Ready for Validation
Sequence Capture
Illumina
TruSeq Exome Enrichment
In Development
Fragment Library Prep
Ion Torrent
Ion Fragment Library Kit
In Development
Fragment Library Prep
Pacific Biosciences
SMRTbellTM Fragment Library Prep
In Development
mRNA Seq
454
cDNA Rapid Library Prep
In Development
Small RNA Seq
Life Technologies
SOLiD Total RNA – Small RNA
In Development
Status as of Feb 2011
65
General NGS Workflow
Caliper Technologies in NGS Workflow
Automation
Sciclone NGS WS and Zephyr NGS WS
Isolate
Sample
Isolation/Purification
DNA and/or RNA
*
Shear
Sample
Generate
Library
Shear/Fragment
Small fragments
Library Generation
- Enzymatic reaction
- Purification
*
Automated Sample Preparation
Quant.
Sample
Sequence
Quantization &
Sample Analysis
Purity and integrity
Sequencing
Seq. chemistry
Microfluidics Analysis
Sequence
Capture
LabChip GX
High Throughput Analysis of DNA and RNA
Microfluidics Preparative
LabChip XT
Automated Nucleic Acid Fractionation and Sizing
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LabChip XT
Automated Size-Selected Fractionation
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LabChip XT – Automated Size-Selected Fractionation
Benefit of LabChip XT
– Quality: Tight and accurate size selection
for sequence assembly, read length &
coverage; Sony manufactured chips
– Data Integrity: Design eliminates crosscontamination
– Cost: Substantial labor savings vs.
manual gels
– Throughput: 96 samples in 12 hrs vs.
weeks
– Speed: 30 min vs. 6 hours
– Reproducibility: Eliminates manual errors
– Safety: No razors or Ethidium Bromide
– Recoverability: Retrieve sample from
collection well and waste well
– Multiple Fractionation
– 4 completely independent channels per
chip
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Workflow
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XT DNA HiRes Assay
Assay Kits
• Available: DNA 750 (50bp to 750bp/1000bp)
• May: DNA 300 (25bp to 500bp)
• 1st Half of 2012: 2 kb and 8 kb Chips
• further in pipeline
High Quality XT DNA Chip manufactured in partnership with Sony DADC
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LabChip XT: Example from Real Life
TGAC, Norwich/UK
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LabChip XT versus SPRI Beads
298bp +/- 12bp (4%)
XT Fractionated Sample
SPRI bead purification yields a resolution of 100 to 200 bp. LC XT below 10% width (e.g. 298 bp ±12 bp or ±4%)
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General NGS Workflow
Caliper Technologies in NGS Workflow
Automation
Sciclone NGS WS and Zephyr NGS WS
Isolate
Sample
Isolation/Purification
DNA and/or RNA
*
Shear
Sample
Generate
Library
Shear/Fragment
Small fragments
Library Generation
- Enzymatic reaction
- Purification
*
Automated Sample Preparation
Quant.
Sample
Sequence
Quantization &
Sample Analysis
Purity and integrity
Sequencing
Seq. chemistry
Microfluidics Analysis
Sequence
Capture
LabChip GX
High Throughput Analysis of DNA and RNA
Microfluidics Preparative
LabChip XT
Automated Nucleic Acid Fractionation and Sizing
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LabChip GX
High Throughput Analysis of DNA and RNA
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LabChipGX features
LabChipGX System
Computer w/ LabChipGX
Software
Reagent Kits
•
•
•
•
•
•
•
•

DNA 1K

DNA 5K

DNA 12K

High Sensitivity DNA

HT RNA
Quantitative sizing and concentration
96 and 384-well compatible
30 to 80 second sample processing
Hit picking capable
Automation compatible
Integrated bar-code reader for sample tracking
21 CFR part 11 compliant software
On instrument chip priming (10 minute setup)
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Caliper LS and Agilent Bioanalyzer
Bioanalyzer 2100
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Liquid Handling Workflow: TrueSeq small RNA Library
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Summary
• Caliper offers with the LabChip XT a low cost solution for
automated fractionation from fragmented DNA reducing labor
and increasing accuracy.
• Based on the same technology, Caliper LabChip GX allows a
significantly higher throughput in NGS sample QC than
Agilent’s Bioanalyzer. The LabChip GX is ideal for highly
parallel sample preparation procedures (e.g. multiplex
sequencing)
• The Caliper Sciclone NGS Workstation is a standalone solution for automated liquid handling in different
applications in NGS sample preparation.
• The integration of all three products (NGS Suite) allows
complete automated solutions for NGS sample preparation.
Lower failure rates in NGS due to more robust and accurate
sample preparation will help to reduce costs significantly.
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Thank you!
Questions?
Contacts:
young.yun@perkinelmer.com
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