The evolution of DNA Sequencing (before Next Gen)

Transcription

1 PerkinElmer Solutions for NGS sample preparation Paul Butler World of Technology and Science, Utrecht PerkinElmer

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3 The evolution of DNA Sequencing (before Next Gen) Slab Gel Capillary Sequencing Auto Radiography Fluorescent 3

4 How many of these = throughput of one of these 60,000 1X10 6 Bases per day 60X10 9 Bases per day 4

5 Overview of PerkinElmer portfolio of genomic and NGS Solutions Our complete portfolio of sample prep solutions upstream of sequencing-low, medium and high throughput New product for library QC LabChip GX Touch 24 Our dedicated NGS post sale support Our library prep automation offer Any chemistry and all sequencer platforms Wide range of library prep protocols Future development roadmap Protocols validated via proof data from the sequencer and documentation Our user community-over 180 automated suites in use for NGS 5

6 PerkinElmer Product Portfolio for NGS Workflows LIMS Informatics - Visualization Isolate Sample Fragment & Sizing Generate Library Quantify Sample Sequence Analyze Prepito / chemagic 360 / JANUS chemagic Nucleic Acid Extraction LabChip XT Automated DNA Fractionation and Sizing LabChip GX Touch Bioanalyzer-like QC for DNA and RNA 6 For Research Use Only. Not For Use in Diagnostic Procedures Sciclone NGS Workstation Automated High-throughput Sequencing Sample Preparation Zephyr NGS Workstation Post PCR Workstation & Sample Preparation NGS Express Benchtop Sequencer Sample Preparation

7 7 PerkinElmer loves the Agilent 2100 Bioanalyzer but!

8 New product -LabChip GX Touch 24 System for fragment size QC System features: Quantification & size QC of NGS libraries, fragments and PCR products Same chip technology as Bioanalyzer with significantly lower cost per sample New 1-24 sample batch size option Multiple assay kits supporting analysis of 25 bp to 12kb fragments including Genomic DNA integrity Fast seconds per sample Walk-away automation of sample batches of 1-24, 1-96 and For Research Use Only. Not For Use in Diagnostic Procedures Full Featured Automation

9 LabChip GX Touch Process Flow Steps: Prepare the Chip Load the Chip Insert Sample Plate Run Automated Assay View Results in Minutes The logical next step for Bioanalyser users! 9 For Research Use Only. Not For Use in Diagnostic Procedures Enhanced Ease of Use

10 Genomic DNA quality-egrams of Degraded vs. Intact gdna Intact λ-dna Sample 2 Sample 3 Sample 4 Degraded Sample For Research Use Only. Not For Use in Diagnostic Procedures

11 Example GQS Scores Scale from 0 (degraded) to 5 (intact) Highest score: 4.1 (λ-dna) Lowest score: 1.0 (Sample 5) 4.1 λ-dna Sample 2 Sample 3 Sample 4 Sample For Research Use Only. Not For Use in Diagnostic Procedures

12 Sequencing Sample Prep Automation NGS Express Zephyr NGS Sciclone NGSx Sample Capacity Key Applications Ideally for... Up to 24 samples per day 8 to 96 samples per day 3 to 5 batches each with 8 to 96 samples per week (depending on application) - Library Prep (MiSeq, PGM, GS FLX) - DNA normalization and pooling - Ion Amplicon Prep - MiSeq Nextera and Amplicon Prep Labs with small bench top NGS systems and moderate weekly thoughput Post PCR - SPRI bead purification - Normalization & qpcr setup - Library Prep (DNA, RNA) - Targeted resequencing Workflows requiring seperation of pre and post PCR processes or for medium/low throughput library prep - Library preparation (DNA, RNA) - Sequence capture - Normalization & pooling - Custom protocols HT sample preparation (e.g. For HiSeq 2000) 12 LOW Throughput Libraries/Application HIGH

13 Informatics/integromicsintegromics and LIMS Sample Tracking Isolate Sample Fragment & Sizing Generate Library Quantify Sample Sequence Analyze Sequencers, applications, and chemistries: so many choices And the list keeps growing Don t get locked in

14 Automation of Illumina chemistries 14

15 Automation of Illumina chemistries User guides Nextera Library prep TruSeq stranded RNA TruSeq DNA TruSeq RNA Nextera on NGS Express Future applications development plan 15

16 Automation of nearly all chemistries! 16 Ion Torrent AmpliSeq PacBio Kapa HTP DNA NimbleGen SeqCap EZ Agilent SureSelectXT NEB

17 Automated library preparation system for Ion AmpliSeq technology by NGS Express LifeTechnologies Japan and Perkin Elmer Japan Introduction Ion AmpliSeq technology is a simple, efficient and rapid process for enriching hundreds to thousands of genomic targets for next generation sequencing from 10 ng of FFPE or whole genomic DNA in a single tube. The flexible nature of this technology scales to meet the demands of both basic and translational research. Although library preparation by manual handling is acceptable for small scale, the trend towards larger sequencing projects involving hundreds of samples presents a unique set of challenges with respect to throughput, cost and uniformity. These problems are compounded when involving associated processes such as sample multiplexing. To avoid errors in library preparation lead to loss of precious samples and wasted reagents. Consider automation to achieve more uniform library preparations. With automation, it can increase the quality of NGS libraries and lower overall costs, while eliminating tedious repetitive procedures. NGS Express Automated Liquid Handling Workstation is a compact, flexible and easy-to-use library prep system for benchtop sequencers such as the Ion Torrent PGM system. It is designed for efficient sample preparation of up to 24 NGS libraries, and eliminates the need for timeconsuming protocol development. Now we are development for automated library preparation for Ion AmpliSeq technology by NGS Express Automated Liquid Handling Workstation. This systems will accelerate sample throughput for Ion AmpliSeq technology without handling error. NGS Express Workstation Ion AmpliSeq Technology Specification Targets Ampliconlength Primer pool size Input DNA required Sample to variants in a single day Up to 5Mb of just your genes or genomic regions 150 bp(optimized for FFPE) or 200 bp(standard) amplicons 24 to 6144 primer pairs per pool 10 ngper primer pool Workflow for Ion AmpliSeq Work with NGS Express + Result Sequence summary Total base: 422M, Total reads: 3,618,544 Total Sample aligned name base to NA17828 hg19: 415M NA12762 MDA-MB-231 SK-MEL-28 Replicate number Mapped Reads 439, , , , , , , ,692 Coverage at 1x % % % % 98.84% 98.84% % % Coverage at 100x 98.86% 99.61% 99.61% 99.08% 97.93% 98.45% 98.86% 98.51% Coverage at 500x 93.22% 97.35% 96.72% 95.99% 94.43% 94.94% 95.71% 92.54% Uniformity of coverage 97.01% 98.81% 97.43% 97.10% 95.51% 97.64% 97.09% 96.55% MDA-MB-231 has deleted part of CDKN2A gene (c.1_471del471, COSMIC ID: COSS934536). Two amplicons in Cancer HP v2 failed to amplify. Confirm with known mutation MDA-MB-231 Muation rate Gene AA Mutation CDS Mutation 1st 2nd TP53 p.r280k c.839g>a BRAF p.g464v c.1391g>t KRAS p.g13d c.38g>a SK-MEL-28 Muation rate Gene AA Mutation CDS Mutation 1st 2nd BRAF p.v600e c.1799t>a EGFR p.p753s c.2257c>t Comparison of mutation rate between replicate 17 Material and Method Samples Reference control NA12878, NA12762 Cancel cell MDA-MB-231, SK-MEL-28 (NCI-60 cell line) Name Tissue Number of KnownMutation In CancerHP v2 MDA-MB-231 Breast 3 SK-MEL-28 Skin 2 Primer pool Ion AmpliSeq Cancer Hotspot Panel v2 Runs 8 barcoded run by 318 CHIP Each sample run duplicate For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use Life Technologies Corporation / 2013 PerkinElmer Japan Co., Ltd. All rights reserved. Conclusion N = 47 We are successful to develop the automated library preparation system for Ion AmpliSeq technology by NGS Express Automated Liquid Handling Workstation. An automated protocol for preparing Ion AmpliSeq has been validated for production use. This system up to 24 libraries can be prepared in a single day. The result of mutation profile show high concordance in duplicate samples. It shows this system has enough reproducibility for ultra-multiplex amplicon re-sequence. This system can reduce steps for manual handling on library preparation step. Sequence data can get stable without any complicate procedure.

18 18 NGS Application Roadmap: NGS Express(1-24 libraries) Vendor Reagent Kit Name Application Area Completion Status Beckman Coulter SPRI Purification Miscellaneous Standard,Factory Installed Beckman Coulter SPRI Size Selection Miscellaneous Standard, Factory Installed PerkinElmer Normalization & Pooling Miscellaneous Standard,Factory Installed Agilent HaloPlex Exome/Targeted Capture Option,Field Installable Agilent SureSelectXT2 Exome/Targeted Capture Option,Field Installable Agilent SureSelectStrand-Specific RNA Library Prep RNA Library Preparation Option,Field Installable BiooScientific NEXTflexDNA Sequencing DNA Library Preparation Option,Field Installable Illumina Nextera DNA Library Preparation Gold Standard Illumina NexteraRapid Capture Exome/Targeted Capture Option,Field Installable Illumina NexteraXT DNA Library Preparation Option,Field Installable Illumina TruSeq Amplicon Exome/Targeted Capture Option,Field Installable Illumina TruSeq Nano DNA Library Preparation Option, Field Installable Illumina TruSeq PCR-Free DNA Library Preparation Option, Field Installable Illumina TruSeq RNA RNA Library Preparation Option, Field Installable Illumina TruSight Tumor Exome/Targeted Capture Option, Field Installable KAPA Biosystems SYBR FAST qpcr Kit Quantification Gold Standard KAPA Biosystems HTP Library preparation DNA Library Preparation Option,Field Installable Life Technologies (Ion Torrent) Ampliseq Panels Exome/Targeted Capture Gold Standard Life Technologies (Ion Torrent) Ion Total RNA Kit v2.0 RNA Library Preparation Option, Field Installable (1-12) New England BioLabsInc. NEBNextUltra DNA Library Preparation Option,Field Installable Roche NimbleGen NimblegenSeqCapEZ Exome/Targeted Capture Option,Field Installable Pacific BioSciences 2kb Template Preparation & Sequencing DNA Library Preparation Option, Field Installable Pacific BioSciences 10kb Template Preparation & Sequencing DNA Library Preparation Option, Field Installable KAPA Biosystems Hyper Prep Kit DNA Library Preparation In development Life Technologies (Invitrogen) PicoGreen Quantification In development

19 NGS Roadmap: Sciclone NGS/NGSx(1-96 libraries) 19 Application Area Reagent Kit Name Sciclone NGS/NGSx DNA Library Preparation Illumina TruSeq DNA Gold Standard (8-96): NOTE ILLUMINA HAS DISCONTINUED DNA Library Preparation Kapa Bio HTP Library Prep Gold Standard (8-96) DNA Library Preparation PacBio DNA Library (10kb) Gold Standard (8-96) DNA Library Preparation Illumina Nextera XT Factory Installable (8-96) DNA Library Preparation NEBNext Factory Installable (8-96) DNA Library Preparation PacBio DNA Library (2kb) Factory Installable (8-96) DNA Library Preparation Illumina Nextera Factory Installable (8-96) DNA Library Preparation Ion Torrent Fragment Library Prep Factory Installable (8-96) DNA Library Preparation Nextera Rapid Capture Factory Installable (8-96) DNA Library Preparation Illumina TruSeq PCR Free Field Verified (8-96) DNA Library Preparation NuGen Encore 3rd Party Installed (8-96) DNA Library Preparation Illumina TruSeq Nano In Development (8-96) Epigenetic Research Custom Chromatin IP Factory Installable (8-96) Exome/Targeted Capture Agilent SureSelect XT Gold Standard (8-96) Exome/Targeted Capture Nimblegen SeqCapEZ User Guide.pdf Gold Standard (8-96) Exome/Targeted Capture Illumina TruSeq Exome Factory Installable (8-96): NOTE ILLUMINA HAS DISCONTINUED Exome/Targeted Capture Agilent HaloPlex Factory Installable (8-96) Exome/Targeted Capture Agilent SureSelect XT2 Factory Installable (8-96) Exome/Targeted Capture Ion Torrent Ampliseq In Development (8-96) Quantification KAPA Bio qpcr Factory Installable (8-32) Quantification PicoGreen Quantitation Factory Installable (8-96) RNAseq Illumina TruSeq RNA Gold Standard (8-96) RNAseq Illumina TruSeq Stranded Total RNA (Ribozero) Gold Standard (8-96) RNAseq Illumina Stranded mrna (poly-a selection) Factory Installable (8-96) RNAseq Epicentre Ribozero/Scriptseq Stranded RNA Field Verified (8-96) RNAseq Illumina Truseq Small RNA Field Verified (8-48) RNAseq NuGen Ovation 3rd Party Installed (8-96) Varied SPRI Purification Factory Installable (8-96) Varied SPRI Size Selection Factory Installable (8-96) Varied Normalization & Pooling Factory Installable (8-96)

20 NGS Roadmap: Zephyr NGS Application Area Reagent Kit Name Zephyr NGS Quantification KAPA Bio qpcr Factory Installable (8-32) Varied SPRI Purification Factory Installable (8-96) Varied Normalization & Pooling Factory Installable (8-96) DNA Library Preparation Illumina TruSeq DNA Factory Installable (8-48) DNA Library Preparation NEBNext Ultra DNA Library Prep Field Verified (8-48), DNA Library Preparation KAPA HTP In Development (8-48) DNA Library Preparation NuGEN Ovation Ultralow Library Prep Field Verified (8-48), Exome/Targeted Capture Illumina TruSeq Amplicon Factory Installable (8-96) Quantification PicoGreen Quantitation Factory Installable (8-96) Exome/Targeted Capture Agilent SureSelect XT Field Verified (8-48), Exome/Targeted Capture Ion Torrent Ampliseq v2.0 In Development (8-48) 20

21 PerkinElmer NGS field support resources available in Europe Several general automation specialists 3 Micro-fluidic technical specialists- Veronika Delcheva Leslie Huber Ilse Klein 3 dedicated NGS post sale automation specialists Yuri Yakushko Julien Rouzade Jose Couto 21

22 Automated FFPE DNA Extraction + Genomic DNA Analysis on GX AGBT 13 22

23 Micro-RNA s using LabChip XT-University of Miami

24 University of Miami

25 28 Tomas Westerling, Dana Farber Cancer Institute Boston

26 Tomas Westerling, Dana Farber Cancer Institute Boston A good sample result is where PGR enhancer1 is high vs DNA yield ng Sciclone automated protocol gives best reproducibility + highest PGR enhancer 1 vs DNA yield 29

27 PerkinElmer s Solution to Genomics analysis: OmicsOffice An Integrative Analysis Suite for Transcriptomics data 31

28 So who uses our solutions? Massachusetts General Hospital OICR University of Queensland UMC Groningen Erasmus Rotterdam 33 RINEN OICR Tokyo University Edge BioSystems Children s Mercy Hospital of Kansas City *2 BGI Shenzen University of Miami Johns Hopkins University Yale University Washington University *2 NCGR JGI Los Alamos National Lab NIH (NHLBI) MD Anderson Cancer Center University of Maryland Vanderbilt University Hudson Alpha Institute University of Connecticut Health Center University of Iowa University of Carolina USAMRID The Genome Analysis Centre TGAC Norwich Sequenom Prince Wales Hospital, Chinese University in Hong Kong ELIM Biopharm Complete Genomics Nestle Sanger Centre

29 Dr Manabu Fujie, Ph.D, OIST, Okinawa, Japan Applications and assays Research To understand better the molecular mechanisms of coral biology including decoding the approximately 420-megabase genome of Acropora digitifera using NGS technology. Using Illumina HiSeqs, Roche 454s, and Life Technologies Solid Sciclone NGS Workstation running home brewed exome capture library prep chemistries LabChip GX for library QC 34

30 Dr Belinda Whittle and Yafei Zhang, APF, ANU Canberra Applications and assays Research The Australian Phenomics Facility (APF) is a specialised facility for developing, characterising and archiving mouse models of human disease, in particular Human and mouse exome sequencing NGS Express Workstation for processing of enriched DNA libraries for their exome sequencing pipeline. LabChip GX for library QC Account Manager: Christine Mylo Back to Index 35

31 Dr Gao Yuan,JHU Biomedical Engineering Faculty, Baltimore, USA Applications and assays Research Sciclone NGS Workstation running exome capture library prep Zephyr The genetic and epigenetic basis of brain function and neurological diseases with a focus on schizophrenia, bipolar spectrum disorder and major depression. Nextgeneration sequencing is bringing the dream closer to the reality of transforming biology into quantitative information. Study brain transcriptome during development, using postmortem human brain and derived ips cells science. Use HiSeq2000 and 454 Junior LabChip GX for library QC LabChip XT Back to Index 36 Account Manager: Eric Herr

32 Toumy Guettouche, Ph.D. University of Miami Hussman Institute Applications and assays 37 Research The Center s ultimate aim is to provide the necessary technical tools and methodological know-how to enable a complete systems approach to the study and analysis of human disorders. Such studies include disease gene localization (genotyping), gene expression analysis, modern ultra-high-throughput next generation (NGS) sequencing and re-sequencing, and analysis of the effects of epigenetic abnormalities.. Sciclone and Zephyr Workstations for Targeted re-sequencing Whole exome sequencing (also in CLIA lab) Whole genome sequencing RNAseq (all applications) ChipSeq TruSeq DNA and exome Agilent SureSelect XT LabChip GX for library QC using gdna assay LabChip XT for mirna s Back to Index Account Manager: Eric Herr

33 Example: Detecting Rare Childhood Diseases 38

34 Neonatal Diagnosis by WGS in Two Days!: STAT-Seq Sci Tranl Med 4, 154ra135 (20120) These kinds of innovation will help more hospitals bring sequencing into clinical care, says Richard Gibbs, director of the human genome sequencing centre at Baylor College of Medicine in Houston, Texas. A lot of people are going to realize from this that the future is now. Facilitated by debottlenecking sample prep and informatics

35 Zephyr NGS: Customer Testimonial Dr. Michael A. Quail Sequencing R&D Team Leader The Wellcome Trust Sanger Institute, Hinxton/UK We find the Zephyr NGS easy to use and flexible enabling processing of just one column of samples up to a whole plate. We mostly use it for AMPure purification of post PCR NGS fragment libraries which are recovered in high yield when compared to performing the protocol manually. Capabilities SPRI purification protocol Normalization protocol Pooling Protocol qpcr set up Custom methods 40 Zephyr NGS Workstation

36 Sciclone NGS: Customer Testimonial Dr. Keith Harshman Coordinator, Genomic Technologies Facility Center for Integrative Genomics University of Lausanne PerkinElmer s combination of integrated hardware and software coupled with their excellent technical support made it possible for us to increase our exome library preparation output 10-fold Capabilities Library preparation (DNA, RNA) Sequence Capture Normalization & Pooling Custom methods 41 Sciclone NGS Workstation

37 NGS Automation Suite: Customer Testimonial Jakob Hedegaard M.Sc., Ph.D., Associate Professor Department of Molecular Medicine (MOMA) Aarhus University Hospital, Skejby, Denmark Using the Sciclone NGS, Zephyr NGS and the LabChip GX for our preparation of NGS libraries, has resulted in higher throughput and reproducibility compared to manual library preparation. We find the instruments easy to use and the supplied methods to be flexible in terms of number of samples to process, volumes etc. We have received excellent service and support from PerkinElmer, both during installation and operation. Pre-PCR Post-PCR Sciclone NGS Workstation Zephyr NGS Workstation LabChip GX 42 DNA-Capture-Seq - TruSeq DNA library preparation - Pre-amplification and purification - Pooling for Nimblegen capture - Setting up post-capture PCR RNA-Seq - RiboZero rrna depletion - ScriptSeq library preparation - Setting up amplification reactions - Purification of amplified libraries - KAPA qpcr - Normalization and pooling for RNA-Seq - Custom methods - Library QC

38 Publication by Sequenom (2013) The development presented here was preceeded by research activities and followed by additional verification and validation studies conducted in a CLIA-certified laboratory. The combined semi-automated process produced 96 libraries in 5 hours, requiring only a single technician and 1.5 hours of hands-on labor time. This resulted in a 4-fold increase in throughput coincident with a 4-fold decrease in labor without sacrificing library yield or quality. 43

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40 KEC Case Study Perkin Elmer TGAC is the European Reference Site for Perkin Elmer Suite of Perkin Elmer instrumentation Gold Standard protocols Bespoke methods Enhanced programming Training workshops Visits and Tours Knowledge Exchange and Commercialisation The Genome Analysis 45 The Genome Analysis Centre Centre

41 The Perkin Elmer Suite at TGAC Zephyr with Twister 2 LabChip GX 3 x Sciclone NGS (1 with Twister 2 and MeCour cooling tower) NGS Express workstation LabChip XT 46 The Genome Analysis Centre

42 Application Current automated protocols carried out on Perkin Elmer instrumentation at TGAC: Illumina Truseq RNA Illumina Truseq DNA Illumina Nextera XT NimbleGen SeqCap EZ Target Enrichment Bead based size-selection Library normalisation Library pooling Quality Control assay set up 47 The Genome Analysis Centre

43 BGI Shenzen China LabChip GX for NGS QC 49 6 LabChip GX installed in BGI China, more worldwide

44 Broad Institute: Pico DNA Assay on GX for NGS QC 50

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