Rapid, High-Resolution Microfluidic Separations for ...€¦ · Rapid, High-Resolution Microfluidic Separations for Nucleic Acid Analysis Rick Bunch Portfolio Director, Microfluidics

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



Your Moderator

Tamlyn Oliver Managing Editor

Genetic Engineering & Biotechnology News



Rick Bunch Portfolio Director, Microfluidics

PerkinElmer

4 4 © 2009 PerkinElmer

GEN Webinar

September, 2012

Introduction to LabChip®

GX Assays

© 2011 PerkinElmer

5 5

Agenda

• Microfluidic Technology

• LabChip GX Automated

Electrophoresis System and Assays

• How Does a Chip Work?

6 6

PerkinElmer Microfluidic Technology Overview

7 7

Concept of Microfluidics

• Miniaturization

• Integration

• Automation

Functionalities Sample prep Separations Reactions Amplification Cell analysis

Applications Genomics Proteomics Drug Discovery Molecular Diagnostics Immunodiagnostics

8 8

LabChip Microfabrication Process

Light

Glass or Quartz

Mask

Develop

Etch

Expose

Photolithography and Etching

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)

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

11 11

LabChip GX System High Throughput Nucleic Acid Analysis for Genomics and Diagnostics Applications

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

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


68 seconds per well



HT DNA 12K

100 bp – 12,000 bp


68 seconds per well



HT DNA High Sensitivity

50 bp – 5000 bp

10 pg/uL – 500 pg/uL

68 seconds per well


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 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

14 14

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

15 15

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

16 16

LabChip GX Automated Electrophoresis System How Does a Chip Work?

17 17

Sipper Chip Layout – DNA/RNA Application

Vacuum Well

Sipper

Separation Channel

Marker Well

18 18

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

19 19

Step 2: Cross-Injection

Voltage drives sample across the separation channel

Separation

Channel

20 20

Step 3: Separation

Detection

Point

Sample (in this case, DNA) is separated and detected in the separation channel.

Detection Window

Separation Channel

21 21

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



Toumy Guettouche, Ph.D. Director

Oncogenomics Core Facility

Sylvester Cancer Center

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


University of Miami, School of Medicine

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

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

2nd Generation sequencing applications at the HIHG-CGT:

• Whole exome sequencing

• Targeted re-sequencing

• Whole genome sequencing

• RNAseq (all applications)

• ChipSeq

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)

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

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

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

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

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

LabChip GX vs. BioAnalyzer 2100 vs. Qubit:

DNA concentration measurement-Ex1

ng

/µl

LabChip GX vs. BioAnalyzer 2100:

DNA size measurement - Ex1

basep

air

s

ng

/µl

LabChip GX vs. BioAnalyzer 2100 vs. Qubit:

DNA concentration measurement – Ex2

Bioanalyzer 2100

LabChip GX

LabChip GX vs. BioAnalyzer 2100:

DNA size measurement – Ex2 b

as

ep

air

s

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)

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)

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



Loida Navarro

Yoslayma Cardentey

Kathy Slosek

Corneliu Sologon

THANK YOU!



Esperanza Anguiano

Director

Genomics Core Laboratory Operations

Baylor Institute for Immunology Research



Rapid, High-Resolution Microfluidic Separations for Nucleic Acid

Analysis

Q&A



Your Moderator

Tamlyn Oliver Managing Editor

Genetic Engineering & Biotechnology News



Rick Bunch Portfolio Director, Microfluidics

PerkinElmer



Toumy Guettouche, Ph.D. Director





Esperanza Anguiano

Director

Genomics Core Laboratory Operations

Baylor Institute for Immunology Research



Thank You For Attending

Rapid, High-Resolution Microfluidic Separations for Nucleic Acid

Analysis

Broadcast Date: Wednesday, October 10, 2012

Time: 11 AM ET

Sponsored by

Documents

Rapid, High-Resolution Microfluidic Separations for ...€¦ · Rapid, High-Resolution Microfluidic Separations for Nucleic Acid Analysis Rick Bunch Portfolio Director, Microfluidics