Next-Generation Sequencing: custom solutions

Transcription

1 Next-Generation Sequencing: custom solutions Julien Abriol & Olivier Lucas, Ph.D. Territory Account Manager Inside Sales Consultant Illumina, Inc. All rights reserved. Illumina, illuminadx, Solexa, Making Sense Out of Life, Oligator, Sentrix, GoldenGate, GoldenGate Indexing, DASL, BeadArray, Array of Arrays, Infinium, BeadXpress, VeraCode, IntelliHyb, iselect, CSPro, GenomeStudio, Genetic Energy, HiSeq, HiScan, TruSeq, Eco, MiSeq and Nextera are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.

2 Illumina at a Glance Founded in 1998 Headquarters in San Diego, CA More than 850,000 sq. ft. Facilities in 7 countries Over 2,300 employees >1200 R&D staff >400 support personnel IP portfolio of >235 patents Added to the NASDAQ-100 listing in 2008 Forbes Fastest Growing Technology Company 2007 &

3 Global Organization Expanded Manufacturing, R&D, Sales, Service & Support Illumina BV (The Netherlands) Illumina China (Beijing) Illumina Hayward (Hayward, CA) Illumina Global Headquarters (San Diego, CA) Illumina Cambridge Illumina Brazil Russia Korea Greece Turkey Jinan, China Illumina KK (Tokyo) Israel Chengdu, China Middle East Shanghai India Taiwan Thailand Vietnam Illumina Singapore Malaysia South Africa Commercial Mfg/R&D Partners Over 2300 Employees >1200 R&D staff >400 Support Personnel Australia New Zealand 3

4 Serving Many Customers Consumer Agriculture Cancer BioPharm Jay Flatley President and CEO Genetics Forensics Infectious Disease Research Human Health Reproductive Health 4

5 Our Vision Innovating for the Future of Genetic Analysis From Genome Wide Discovery To Targeted Validation and Beyond To be the leading provider of integrated solutions that advance the understanding of genetics and health 5

6 Illumina Portfolio Overview Innovation is in our DNA From Genome-Wide Discovery to Targeted Validation and Screening Sequencing Arrays qpcr HiSeq 2500 Redefining the trajectory of sequencing HiSeq 1500 Powerful, Flexible, Scalable Genome Analyzer IIx Most widely adopted NGS platform HiScanSQ Unique combination of sequencing and arrays iscan Speed, quality and versatility for arrays BeadXpress Accuracy, versatility and flexibility for molecular testing Eco Gold-standard qpcr made accessible Miseq 6

7 Illumina Genomics Power and flexibility Gene Expression / Regulation GEX Methylation ChIP-Seq Small RNA ChIP-Seq DNA Analysis Whole-genome sequencing Metagenomics Targeted resequencing resequencing CNV de novo sequencing 7

8 Industry New enzymes Natural products Medical Genetic engineering Pharmacogenomics Metagenomics Genetic analysis Agricultural Microalgae Natural products M a r i n e B i o t e c h n o l o g i e s Fisheries Biodiversity Traceability (Barcoding/metagenomics) Acquaculture Genetic selection Feed traceability 8

9 Illumina in marine biology and marine biotechnology Woods Hole Marine Biological Lab now fully transitionned Transcriptomics of microinvertebrates (Welch lab) Marine biodiversity/encyclopedia of Life (Zettler lab) Functional genomics of microbial genomes (Serres lab) Toxicology (Hamilton lab) Metagenomes (Sogin, Huse, Morrisson, Eren labs) Marine Genomics 4 Users (mg4u.eu): «for a targeted transfer of knowledge to Industry Genomics in marine monitoring: New opportunities for assessing marine health status (2013) Bourlat et al. Marine Pollution Bulletin 9

10 Next Generation Sequencing Instruments Illumina, Inc. All rights reserved. Illumina, illuminadx, Solexa, Making Sense Out of Life, Oligator, Sentrix, GoldenGate, GoldenGate Indexing, DASL, BeadArray, Array of Arrays, Infinium, BeadXpress, VeraCode, IntelliHyb, iselect, CSPro, GenomeStudio, Genetic Energy, HiSeq, HiScan, TruSeq, Eco, MiSeq and Nextera are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.

11 Data Equivalence. 60,000 ABI MiSeq HiSeq

12 Of MiSeq v2, Microbes, and Man Organism Genome size n depth Staphylococcus aureus (MRSA) 2.8 Mb 27 50x Mycobacterium tuberculosis (TB) 4.4 Mb 18 50x Escherichia coli 4.6 Mb 18 50x Plasmodium falciparum 22.9 Mb 6 30x Human Target size n depth 20 exons 3 kb x Targeted region 0.5 Mb x All coding exons 25 Mb 3 40x RNA, mirna, ChIP-Seq, etc 6M tags 3 n.a. 12

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14 Workflow Illumina, Inc. All rights reserved. Illumina, illuminadx, Solexa, Making Sense Out of Life, Oligator, Sentrix, GoldenGate, GoldenGate Indexing, DASL, BeadArray, Array of Arrays, Infinium, BeadXpress, VeraCode, IntelliHyb, iselect, CSPro, GenomeStudio, Genetic Energy, HiSeq, HiScan, TruSeq, Eco, MiSeq and Nextera are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.

15 Illumina Sequencing Workflow 3 5 DNA ( µg) Library Preparation Single molecule array Cluster Growth A T C A C C G T G G A G C 5 T G T A C G A T C A C C C G A T C G A A Sequencing T G T A C G A T Image Acquisition Base Calling 15

16 16 MiSeq System

17 MiSeq Single Instrument Workflow The World s Most Widely Adopted Sequencing Technology Just Got Personal 2007: 2010: 2011: GA MiSeq HiSeq launch, launch, the year 3 NGS genomes/run of in the routine 1 st Gb Included On-Instrument: Cluster Generation Paired-End Fluidics Computing for Primary and Secondary Analysis 17

18 Illumina Sequencing Workflow 1 Library Preparation Fragment DNA Repair ends Add A overhang Ligate adapters Purify 2 Cluster Generation Hybridize to flow cell Extend hybridized template Perform bridge amplification Prepare flow cell for sequencing 3 Sequencing Perform sequencing Generate base calls 4 Data Analysis Images Intensities Reads Alignments 18

19 MiSeq: for all you seek Integrated. Optimized. Simplified. Amplicon Sequencing Custom Amplicon Targeted Resequencing Custom Enrichment Small RNA sequencing Clone checking ChIP-Seq Library QC Plasmid Regulation RNA-Seq Resequencing Small genome RNA sequencing De novo sequencing 16S Metagenomics 19

20 MiSeq Continuous Performance Improvements Path towards 15Gb per run; enabling broader range of applications 20 Output Clusters Read length 15 Gb 25M 2 x 300 bp Output >8 Gb Clusters >15M Output - Gb 10 Output >1.5 Gb Clusters ~7M Read length 2 x 150 bp Read length 2 x 250 bp August Faster chemistry Dual surface imaging 2Q11 3Q11 4Q11 1Q12 2Q12 3Q12 4Q12 1Q13 2Q13 3Q13 4Q13 This information is intended to outline general product direction and it should not be relied on in making a purchasing decision. This material is for information 20 purposes only and may not be incorporated into any contract. This information is not a commitment, promise, or legal obligation to deliver this functionality. The development, release, and timing of any features or functionality described for our products remains at our sole discretion.

21 MiSeq Continuous Performance Improvements Path towards 15Gb per run; enabling broader range of applications 21

22 22 HiSeq 2500 Sequencing System

23 HiSeq 2500 System Combining innovation MiSeq HiSeq 2500 HiSeq 2000 Clustering on-board Fast Chemistry Longer Reads Rapid turnaround Clustering on-board Complete walk-away workflow Longer 2x150 reads Data rate TDI scanning Larger flow cell 23

24 HiSeq 2500 Sequencing System Fast turnaround and highest output in a single instrument 1 Instrument 2 Run Modes High Output Mode 600 Gb in ~10.5 days 3 billion clusters cbot required Rapid Run Mode 120Gb in ~1 day 600 million clusters No cbot required User configurable 5 human genomes in 10.5 days 1 human genome in a day Highest output Fastest turnaround 24

25 Acquisition of Moleculo Enabling synthetic read lengths up to 10Kb from Illumina short reads Proprietary technology for phased synthetic long reads (>10Kb) from short reads Novel library prep & analysis algorithm Complete solution including sample prep, sequencing w/ Illumina systems, cloud-based informatics Obviates need for long read systems High accuracy from Illumina SBS Anticipated availability Services already available! Commercial kits Q

26 26 Applications

27 Industry New enzymes Natural products Medical Genetic engineering Pharmacogenomics cs Metagenomics MiSeq/ HiSeq Agricultural Microalgae Natural products M a r i n e B i o t e c h n o l o g i e s Fisheries Biodiversity Traceability (Barcoding/metagenomics) ics) Acquaculture Genetic selection Feed traceability 27

28 NexteraXT DNA Sample Preparation Kit Sequencing s fastest and easiest sample prep From DNA to sequencing-ready library in as little as 90min 2011 Illumina, Inc. All rights reserved. Illumina, illuminadx, Solexa, Making Sense Out of Life, Oligator, Sentrix, GoldenGate, GoldenGate Indexing, DASL, BeadArray, Array of Arrays, Infinium, BeadXpress, VeraCode, IntelliHyb, iselect, CSPro, GenomeStudio, Genetic Energy, HiSeq, HiScan, TruSeq, Eco, MiSeq and Nextera are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.

29 Step 1: Tagmentation of template DNA 29

30 Step 2: PCR to add adapters and indices 30

31 Step 3: Cleanup and Sequence 31

32 Sample Normalization is included No library quantification or qpcr is required go straight to MiSeq! Completed libraries, range of yields Index CV for 20-sample pools Pool A Pool B Quant in triplicate with qpcr Calculate dilutions Manually dilute and pool 15.8% 18.2% 5 µl of each desired library Bead-based Normalization: Bind, Wash, Elute 13.5% 15.5% Nextera XT sample pooling is as simple as pipetting 5 µl! 32

33 Streamlined Library Preparation 100ng-5 µg ~4-6 hours 1 or 50 ng <2 hours Nexter a 33

34 34 Microbial profiling with 16S

35 16S ribosomal DNA for microbial profiling V1 V2 V3 V4 V5 V6 V7 V8 V9 C1 C2 C3 C4 C5 C6 C7 C8 C9 16S rrna forms part of bacterial ribosomes. Contains regions of highly conserved and highly variable sequence. Large public 16S databases available for comparison Conserved regions can be targeted to amplify broad range of bacteria from environmental samples. Not quantitative due to copy number variation 35

36 16S rrna Metagenomics 36

37 37 Small genome (re)sequencing

38 MiSeq Applications Small Genome Resequencing Resequencing of 5.2Mb B. cereus in a single workday 5.4 million reads yielded 175Mb of data which aligned to ATCC10987 with mismatch rate of 0.06% >98% of genome with average coverage of 30x gdna Prep Library 1.5 hours (15-30 min hands on) Clustering + Sequencing 4.5 hours (20 mins hands on) Align to reference/call SNPs 2 hours On Board Instrument 38 *1x36bp

39 MiSeq Applications Small Genome Resequencing Resequencing of 3 algae strains Genome size: 50Mb gdna Prep Library 1.5 hours (15-30 min hands on) Desired coverage 30X 4.5Gb of sequencing (2x150bp) Indicative costs: 4*40= /genome Clustering + Sequencing 24 hours (20 mins hands on) Align to reference/call SNPs >2 hours On Board Instrument 39

40 RNA seq For 40 Research Use Only

41 RNAseq adoption Publications in Pubmed relative to RNAseq or microarrays (September 30th, 2013; normalized to max) Data pulled from NIH database, represents grants in 1 st year of support Mortazavi et al., (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature Methods 41

42 RNA Seq Description Quantitate levels of RNA expression Discover and profile mrna in any eukaryotic species Obtain full sequence from any poly-a tailed RNA to analyze novel transcripts, novel isoforms, alternative splice sites, rare transcripts, and csnps in one experiment. Profile small RNA in any organism without any prior assumptions Find novel micrornas, characterize mutations, and analyze the differential expression of all small RNAs Generate reads that maintain information about the strandedness of the transcript for transcriptome annotation or bacterial transcriptome profiling Discover and profile non-coding RNA (ncrna) in the transcriptome 42

43 Experimental Design Required Read depth Required Reads Per Sample for RNA-Seq (Human) 43

44 TruSeq RNA Workflow Overview mrna kit Total RNA kit 44

45 RNA-Seq Benefits A Sequencing-based Technology to Profile the Transcriptome Qualitative and quantitative RNA analysis Any species - even when reference genome not available No prior knowledge required Alternative transcripts Gene fusions csnps, allele-specific expression 45

46 Illumina s Suite of Sample Prep Solutions DNA Sample Prep Nextera Nextera XT TruSeq DNA LT & HT TruSeq DNA PCR-free Nextera Mate Pair Targeted Resequencing TruSeq Custom Amplicon TruSeq Exome & Custom Enrichment Nextera Exome & Custom Enrichment RNA & Regulation Sample Prep TruSeq Small RNA TruSeq RNA TruSeq Stranded RNA (FFPE) TruSeq ChIP 46

47 47 Thank You!

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