rapid, high-resolution microfluidic separations for ...€¦ · rapid, high-resolution microfluidic...
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Rapid, High-Resolution Microfluidic
Separations for Nucleic Acid Analysis
Broadcast Date: Wednesday, October 10, 2012
Time: 11 AM ET
Sponsored by
Rapid, High-Resolution Microfluidic Separations
for Nucleic Acid Analysis
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Rapid, High-Resolution Microfluidic
Separations for Nucleic Acid Analysis
Your Moderator
Tamlyn Oliver Managing Editor
Genetic Engineering & Biotechnology News
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Rapid, High-Resolution Microfluidic
Separations for Nucleic Acid Analysis
Rick Bunch Portfolio Director, Microfluidics
PerkinElmer
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4 4 © 2009 PerkinElmer
GEN Webinar
September, 2012
Introduction to LabChip®
GX Assays
© 2011 PerkinElmer
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5 5
Agenda
• Microfluidic Technology
• LabChip GX Automated
Electrophoresis System and Assays
• How Does a Chip Work?
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6 6
PerkinElmer Microfluidic Technology Overview
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Concept of Microfluidics
• Miniaturization
• Integration
• Automation
Functionalities Sample prep Separations Reactions Amplification Cell analysis
Applications Genomics Proteomics Drug Discovery Molecular Diagnostics Immunodiagnostics
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8 8
LabChip Microfabrication Process
Light
Glass or Quartz
Mask
Develop
Etch
Expose
Photolithography and Etching
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9 9
LabChip Formats High Throughput and Personal Scale
Sipper Chip Automated sampling from microtiter plates for high throughput (LabChip EZ Reader, LabChip GX)
Planar Chip Manual reagent introduction for low sample number, disposable (Agilent, Biorad)
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10 10
PerkinElmer Microfluidic Platforms
Enzyme Activity Assays
Compound Profiling
Mechanism of Action
Kinetic capability
DNA/RNA
Protein/Glycans
High speed Micro-CE
Preparative DNA
Electrophoresis
Fractionation/collection
LabChip EZ Reader for Enzymatic Assays
and Selectivity Profiling
LabChip GX & GXII Automated
Electrophoresis Systems
LabChip XT Nucleic Acid
Fractionation System
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11 11
LabChip GX System High Throughput Nucleic Acid Analysis for Genomics and Diagnostics Applications
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12 12
Overview of LabChip GX System
Instrument Kits Software
Automated 1-D electrophoretic separations of RNA and DNA
Capillary electrophoresis performed in microfluidic chips
Sample directly from 96- or 384-well microtiter plate
Comprehensive and quantitative analysis with LabChip GX software Optional 21 CFR Part 11 compliance
package
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13 13
HT DNA 5K
100 bp – 5000 bp
0.25 ng/uL – 50 ng/uL per fragment
28 seconds per well
45 min run time for 96-well plate
Chip and reagents for 2000 samples
HT DNA 1K
25 bp – 1000 bp
0.1 ng/uL – 50 ng/uL per fragment
68 seconds per well
120 min run time for 96-well plate
Chip and reagents for 2000 samples
HT DNA 12K
100 bp – 12,000 bp
0.25 ng/uL – 50 ng/uL per fragment
68 seconds per well
120 min run time for 96-well plate
Chip and reagents for 2000 samples
HT DNA High Sensitivity
50 bp – 5000 bp
10 pg/uL – 500 pg/uL
68 seconds per well
120 min run time for 96-well plate
Chip good for 2000 samples
Reagents for 400 samples
HT RNA
100 nt – 6000 nt
5 ng/uL - 250 ng/uL
80 seconds per well
2.5 hour run time for 96-well plate
Chip and reagents for 1000 samples
Chip and reagents sold separately
pico RNA
100 nt – 6000 nt
500 pg/uL - 5000 pg/uL
80 seconds per well
2.5 hour run time for 96-well plate
Supports up to 500 samples per reagent kit
Chip and reagents sold separately
LabChip GX Nucleic Acid Kits
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Protocol Flexibility of Kits
Kit Name High Throughput
Protocol
Small Batch
Protocol
High Sens DNA
DNA 1K
Pico RNA
DNA 5K
DNA 12K
Standard RNA
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Sample detection via laser-induced fluorescence
Excitation = 635 nm Emission = 700 nm
Single Sipper Chip RFID tag stores chip identity
Applications PCR Analysis & QC Genotyping Restriction digest analysis RFLP/AFLP DNA Diagnostics Smear Analysis for NGS
DNA Analysis on the LabChip GX System
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16 16
LabChip GX Automated Electrophoresis System How Does a Chip Work?
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Sipper Chip Layout – DNA/RNA Application
Vacuum Well
Sipper
Separation Channel
Marker Well
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Step 1: Sampling
Vacuum is applied to the Vacuum Well.
Markers flow toward the Vacuum Well
Sample flows through the sipper and mixes with the Markers
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Step 2: Cross-Injection
Voltage drives sample across the separation channel
Separation
Channel
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Step 3: Separation
Detection
Point
Sample (in this case, DNA) is separated and detected in the separation channel.
Detection Window
Separation Channel
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LabChip GX for NGS
• High throughput library sizing and quantification
• Advanced smear analysis
• 96 samples in 2 hours
• Automation capable
• Integrated sample tracking
• Digital data; LIMS compatible
Use after shearing and/or after library construction
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Rapid, High-Resolution Microfluidic
Separations for Nucleic Acid Analysis
Toumy Guettouche, Ph.D. Director
Oncogenomics Core Facility
Sylvester Cancer Center
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The LabChip GX in a Genomics Core
Facility
Toumy Guettouche, Ph.D.
Assistant Professor, Hussman Institute for Human Genomics,
Dr. John T. MacDonald Foundation Department of Human Genetics
Director Genomics Technology Assessment and Implementation, Center for
Genome Technology; Core Director, Oncogenomics Core Facility
Sylvester Cancer Center
University of Miami, School of Medicine
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Center for Genome
Technology (CGT)
Oncogenomics Core
Facility (OCF)
University of Miami, Miller School of Medicine,
Genomics Core Structure
Biomedical
Research Building
(opened July 2009)
• Genotyping
• Gene Expression
• Capillary Sequencing
• 2nd Generation Sequencing
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Age-related macular degeneration (AMD)
Alzheimer disease
Amyotrophic lateral sclerosis (ALS)
Asperger disorder
Autism
Charcot-Marie-Tooth disease (CMT)
Cancer
Deafness
Hereditary Spastic Paraplegia (HSP)
Multiple sclerosis (MS)
Neural tube defects (NTD)
Parkinson disease (PD) Thrombotic Storm
Tuberculosis (TB)
Trichotillomania (TTM)
Retinitis Pigmentosum
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2nd Generation sequencing applications at the HIHG-CGT:
• Whole exome sequencing
• Targeted re-sequencing
• Whole genome sequencing
• RNAseq (all applications)
• ChipSeq
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Quality Control of Genomics Applications :
• RNA quality Control (“RIN”) from all sources including FFPE
• RNAseq QC, library quant (except Ribozero method)
• Microarray QC (cRNA)
• DNA Quality Control including FFPE
• DNAseq QC (Exomes, WGS, targeted, ChipSeq)
• PCR products
• (Genomic DNA)
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Basic Workflow of Bioanalyzer 2100 and LabChip GX
Reagent Prep
Prime Chip
Vortex Chip
Load Samples
Run Chip
Reagent Prep
Prep & Setup Chip
Prep Sample Plate
Load Chip&Plate
Run Chip
Buffer tube
Ladder tube
Sample plate
Bioanalyzer 2100 LabChip GX
Load Chip
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Setup/Priming
Run Samples (12)
Analyze data
Setup/Priming
Run Samples (96)
Analyze data
Duration
10-20 min
30 min
10-30min
Duration
30 min
120 min
10-30 min
x 8 for 96 samples x 1 for 96 samples
Total Time required for 96 samples
Total 400 min = 6h 40min 180 min= 3h
Hands on 160 min = 2h 40min 60 min=1h
Agilent BioAnalayzer 2100 PE/Caliper LabChip GX
Comparison of Processing Time for 96 samples
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Day 1
•Setup overnight shearing of sample DNA plate in the Covaris E210
Day 2
•Sheared DNA cleanup using AMPure XP Beads in the Caliper Sciclone G3
•Quality assessment of the sheared DNA in the Caliper LabChip GX
Day 3
•Library preparation:
-End Repair
-Add A-bases
-Ligate adapters
-Amplify ligated library
Day 4
•Quality and quantity assessment in the Caliper LabChip GX
•Normalize library for hybridization
Day 5
•Set-up of 24-hour library hybridization to capture probes
Day 6
•Recovery of captured DNA
•Amplification of captured DNA
•Setup of overnight quality and quantity assessment of final product
Day 7
•Submission of final product to sequencing core.
Exome/Targeted Capture Workflow
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Workflow Summary
Shear DNA
SPRI Clean Up
Library Preparation
Enrichment PCR
SPRI Clean Up
Normalization
Hybridization
Target Selection
Amplification (non-indexing or indexing)
SPRI Clean Up
QC with GX
QC with GX
Incubation
Thermocycler
QC with GX
Onlin
e P
rocesses
Offlin
e P
rocesses
Pre PCR
Post PCR
Sciclone G3
NGS
Zephyr
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Total Time required for 3x96 samples
Bioanalyzer 2100 LabChip GX
Total 1200 min = 20h 540 min= 9h
Hands on 480 min = 8h 180 min= 3h
Time saved for Exome Capture QC using
LabChip GX:
• 11h Processing time
• 5h Hands on time
LabChip GX vs. BioAnalyzer 2100: Total Processing time for
Exome/Targeted Capture QC
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LabChip GX vs. BioAnalyzer 2100 vs. Qubit:
DNA concentration measurement-Ex1
ng
/µl
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LabChip GX vs. BioAnalyzer 2100:
DNA size measurement - Ex1
basep
air
s
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ng
/µl
LabChip GX vs. BioAnalyzer 2100 vs. Qubit:
DNA concentration measurement – Ex2
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Bioanalyzer 2100
LabChip GX
LabChip GX vs. BioAnalyzer 2100:
DNA size measurement – Ex2 b
as
ep
air
s
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Cervix
TPS Method A
*
TPS Method A
Bladder
*
TPS Method A
Kidney
*
Caliper GX simulated gel picture showing the molecular weight of DNA extracted from different tumor tissues using a commercially available manual FFPE extraction method (Method A) and the Siemens TPS. 2ng of DNA were loaded per sample. Results from 6 consecutive sections are shown. *designates high molecular weight DNA (above 7 kilobases).
FFPE DNA and RNA extraction (CLIA service)
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Summary:
LabChip GX Positives
• Throughput
• Cost per data Point
• Analysis Software
LabChip GX Negatives
• RNA size starts at 100bp vs 20bp on BioA
• (minimum size of kits)
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Caliper/PE
Nate Cosper
Isaac Meek
Eric Herr
Agilent
Matthew Angel
Victor Miller
Hussman Institute for Human
Genomics
Center for Genome Technology
Research and Development
Joe Rantus
Sequencing Team
Bill Hulme
Ashley Andersen
Ashley Diaz
Ryan Gentry
Capture Team
Anna Konidari
Christina Siebert
Anat Aviram
Yuslin Pasco
Rosanna Tursi
Eminisha Lalanne
Natalie Leyva
Sylvester Cancer Center
Oncogenomics Core Facility
Loida Navarro
Yoslayma Cardentey
Kathy Slosek
Corneliu Sologon
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THANK YOU!
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Rapid, High-Resolution Microfluidic
Separations for Nucleic Acid Analysis
Esperanza Anguiano
Director
Genomics Core Laboratory Operations
Baylor Institute for Immunology Research
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Rapid, High-Resolution Microfluidic
Separations for Nucleic Acid Analysis
Rapid, High-Resolution Microfluidic Separations for Nucleic Acid
Analysis
Q&A
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Rapid, High-Resolution Microfluidic
Separations for Nucleic Acid Analysis
Your Moderator
Tamlyn Oliver Managing Editor
Genetic Engineering & Biotechnology News
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Rapid, High-Resolution Microfluidic
Separations for Nucleic Acid Analysis
Rick Bunch Portfolio Director, Microfluidics
PerkinElmer
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Rapid, High-Resolution Microfluidic
Separations for Nucleic Acid Analysis
Toumy Guettouche, Ph.D. Director
Oncogenomics Core Facility
Sylvester Cancer Center
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Rapid, High-Resolution Microfluidic
Separations for Nucleic Acid Analysis
Esperanza Anguiano
Director
Genomics Core Laboratory Operations
Baylor Institute for Immunology Research
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Rapid, High-Resolution Microfluidic
Separations for Nucleic Acid Analysis
Thank You For Attending
Rapid, High-Resolution Microfluidic Separations for Nucleic Acid
Analysis
Broadcast Date: Wednesday, October 10, 2012
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