Rapid, High-Resolution Microfluidic Separations for Nucleic Acid Analysis

Transcription

1 Rapid, High-Resolution Microfluidic Separations for Nucleic Acid Analysis Rapid, High-Resolution Microfluidic Separations for Nucleic Acid Analysis Broadcast Date: Wednesday, October 10, 2012 Time: 11 AM ET Sponsored by

2 Rapid, High-Resolution Microfluidic Separations for Nucleic Acid Analysis Your Moderator Tamlyn Oliver Managing Editor Genetic Engineering & Biotechnology News

3 Rapid, High-Resolution Microfluidic Separations for Nucleic Acid Analysis Rick Bunch Portfolio Director, Microfluidics PerkinElmer

4 Introduction to LabChip GX Assays GEN Webinar September, PerkinElmer

5 Agenda Microfluidic Technology LabChip GX Automated Electrophoresis System and Assays How Does a Chip Work? 5

6 6 PerkinElmer Microfluidic Technology Overview

7 Concept of Microfluidics Miniaturization Integration Automation Functionalities Sample prep Separations Reactions Amplification Cell analysis Applications Genomics Proteomics Drug Discovery Molecular Diagnostics Immunodiagnostics 7

8 LabChip Microfabrication Process Photolithography and Etching Expose Light Mask Glass or Quartz Develop Etch 8

9 LabChip Formats High Throughput and Personal Scale 9 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 PerkinElmer Microfluidic Platforms LabChip EZ Reader for Enzymatic Assays and Selectivity Profiling LabChip GX & GXII Automated Electrophoresis Systems LabChip XT Nucleic Acid Fractionation System Enzyme Activity Assays Compound Profiling Mechanism of Action Kinetic capability DNA/RNA Protein/Glycans High speed Micro-CE Preparative DNA Electrophoresis Fractionation/collection 10

11 LabChip GX System High Throughput Nucleic Acid Analysis for Genomics and Diagnostics Applications 11

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 12

13 LabChip GX Nucleic Acid Kits 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 HT RNA pico RNA 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 100 nt 6000 nt 5 ng/ul 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 100 nt 6000 nt 500 pg/ul 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

14 Protocol Flexibility of Kits Kit Name High Sens DNA DNA 1K Pico RNA DNA 5K DNA 12K Standard RNA High Throughput Protocol Small Batch Protocol 14

15 DNA Analysis on the LabChip GX System 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 15

16 LabChip GX Automated Electrophoresis System How Does a Chip Work? 16

17 Sipper Chip Layout DNA/RNA Application Vacuum Well Separation Channel Marker Well Sipper 17

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 18

19 Step 2: Cross-Injection Separation Channel 19 Voltage drives sample across the separation channel

20 Step 3: Separation Detection Window Detection Point Separation Channel Sample (in this case, DNA) is separated and detected in the separation channel. 20

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 21 Use after shearing and/or after library construction

22 Rapid, High-Resolution Microfluidic Separations for Nucleic Acid Analysis Toumy Guettouche, Ph.D. Director Oncogenomics Core Facility Sylvester Cancer Center

23 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

24 University of Miami, Miller School of Medicine, Genomics Core Structure Center for Genome Technology (CGT) Oncogenomics Core Facility (OCF) Genotyping Gene Expression Capillary Sequencing 2 nd Generation Sequencing Biomedical Research Building (opened July 2009)

25 Age-related macular degeneration (AMD) Amyotrophic lateral sclerosis (ALS) Alzheimer disease Deafness Multiple sclerosis (MS) Cancer Thrombotic Storm Neural tube defects (NTD) Charcot-Marie-Tooth disease (CMT) Tuberculosis (TB) Asperger disorder Trichotillomania (TTM) Autism Parkinson disease (PD) Retinitis Pigmentosum Hereditary Spastic Paraplegia (HSP)

26 2 nd Generation sequencing applications at the HIHG-CGT: Whole exome sequencing Targeted re-sequencing Whole genome sequencing RNAseq (all applications) ChipSeq

27 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)

28 Basic Workflow of Bioanalyzer 2100 and LabChip GX Bioanalyzer 2100 LabChip GX Reagent Prep Reagent Prep Prime Chip Prep Sample Plate Prep & Setup Chip Load Samples Vortex Chip Load Chip Load Chip&Plate Sample plate Buffer tube Ladder tube Run Chip Run Chip

29 Comparison of Processing Time for 96 samples Agilent BioAnalayzer 2100 Duration PE/Caliper LabChip GX Duration Setup/Priming min Setup/Priming 30 min Run Samples (12) 30 min Run Samples (96) 120 min Analyze data 10-30min Analyze data 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

30 Exome/Targeted Capture Workflow 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.

31 Online Processes Workflow Summary Shear DNA Pre PCR Sciclone G3 NGS SPRI Clean Up Library Preparation QC with GX Enrichment PCR SPRI Clean Up Normalization Hybridization Thermocycler QC with GX Incubation Offline Processes Target Selection Amplification (nonindexing or indexing) Zephyr SPRI Clean Up QC with GX Post PCR

32 LabChip GX vs. BioAnalyzer 2100: Total Processing time for Exome/Targeted Capture QC Total Time required for 3x96 samples Bioanalyzer 2100 Total 1200 min = 20h 540 min= 9h Hands on 480 min = 8h 180 min= 3h LabChip GX Time saved for Exome Capture QC using LabChip GX: 11h Processing time 5h Hands on time

33 ng/µl LabChip GX vs. BioAnalyzer 2100 vs. Qubit: DNA concentration measurement-ex1

34 basepairs LabChip GX vs. BioAnalyzer 2100: DNA size measurement - Ex1

35 ng/µl LabChip GX vs. BioAnalyzer 2100 vs. Qubit: DNA concentration measurement Ex2

36 basepairs LabChip GX vs. BioAnalyzer 2100: DNA size measurement Ex2 Bioanalyzer 2100 LabChip GX

37 FFPE DNA and RNA extraction (CLIA service) TPS Method A TPS Method A TPS Method A * * * Kidney Bladder Cervix 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).

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39 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)

40 Hussman Institute for Human Genomics Center for Genome Technology Research and Development Joe Rantus Sequencing Team Bill Hulme Ashley Andersen Ashley Diaz Ryan Gentry Sylvester Cancer Center Oncogenomics Core Facility Loida Navarro Yoslayma Cardentey Kathy Slosek Corneliu Sologon Caliper/PE Nate Cosper Isaac Meek Eric Herr Agilent Matthew Angel Victor Miller Capture Team Anna Konidari Christina Siebert Anat Aviram Yuslin Pasco Rosanna Tursi Eminisha Lalanne Natalie Leyva

41 THANK YOU!

42 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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59 Rapid, High-Resolution Microfluidic Separations for Nucleic Acid Analysis Rapid, High-Resolution Microfluidic Separations for Nucleic Acid Analysis Q&A

60 Rapid, High-Resolution Microfluidic Separations for Nucleic Acid Analysis Your Moderator Tamlyn Oliver Managing Editor Genetic Engineering & Biotechnology News

61 Rapid, High-Resolution Microfluidic Separations for Nucleic Acid Analysis Rick Bunch Portfolio Director, Microfluidics PerkinElmer

62 Rapid, High-Resolution Microfluidic Separations for Nucleic Acid Analysis Toumy Guettouche, Ph.D. Director Oncogenomics Core Facility Sylvester Cancer Center

63 Rapid, High-Resolution Microfluidic Separations for Nucleic Acid Analysis Esperanza Anguiano Director Genomics Core Laboratory Operations Baylor Institute for Immunology Research

64 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 Time: 11 AM ET Sponsored by