NGS超入門 - 愛媛大学

1 2014 Illumina, Inc. All rights , 24sure, BaseSpace, BeadArray, BlueFish, BlueFuse, BlueGnome, cBot, CSPro, CytoChip, DesignStudio, Epicentre, GAIIx,Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, HiSeq X, Infinium, iScan, iSelect, ForenSeq, miseq , MiSeqDx, MiSeqFGx, NeoPrep, Nextera, NextBio, NextSeq, Powered by Illumina, SeqMonitor, SureMDA, TruGenome, TruSeq, TruSight, Understand Your Genome, UYG, VeraCode, verifi, VeriSeq, the pumpkin orange color, and the streaming bases design are trademarks of Illumina, Inc. and/or its affiliate(s) in the and/or other other names, logos, and other trademarks are the property of their respective miseq 2 iSchool 3 DNA .. iSchool 4 ~ iSchool iSchool 5 1 NGS 6 1 NGS 2 NGS 1 NGS 7 1 NGS 1.

2 2. 1 NGS 81. 9 NGS 1,000 1 NGS CE 102006 $2,000 2003 $30 2007 $200 200830X $20 11 $2,000 $1,0001/20,0002014$1,000 2010$10,000 1,000 12 DNA 13 1 / 1 / 1,000 bpx19696 Kb150 bpx26,000,000, TbX XXII IIII1 14 V2 50 cycle50bp2x2515M (Small RNA ) Library QCV2 300 cycle300bp2x15015M NexteraXT PCR TruSeqCustom Amplicon V2 500 cycle500bp2x25015M De novo V3 -150 cycle150bp2x7525M RNA-seq( )

3 Exome V3 600 cycle600bp2x30025M denovo HLA 151. 1 1000 16 1 NGS 1. 2. 1 NGS 172. 18 miseq DNA DNA 8 * *Nextera 36bpx1 ( ) [ ] 08:00:00 AmpliconsClonesgDNA19 MiSeqSequencing WorkflowData AnalysisSequencingCluster GenerationLibrary Preparation20 DNADNA bp PCR DNA 21 Illumina : TruSeqsample preparation kit( [DNA] [RNA] [Small RNA], [ChIP-Seq] ) TruSeq Custom Amplicon(TSCA) kit Nexterakit TruSightkit ( ) qPCR Qubit Picogreen ( ) Bioanalyzer Library Preparation Flowcell DNA Index (.)

4 TruSeqDNA HT (Dual Index) 22 index Index 1 23 MiSeqSequencing WorkflowData AnalysisSequencingCluster GenerationLibrary Preparation24 DNA DNA DNA 5 sample to sequencing primerCluster Generation1. DNA 2. PCR 3. 25 Flow Cell ? 26 Flow CellCluster GenerationFlowcell 27 3 extension Cluster GenerationHybridize Fragment & Extend1. Hybridize sample to flowcell28discard DNA Cluster GenerationDenature Double-stranded DNA1. Hybridize sample to flowcell29 Cluster GenerationDenature Double-stranded DNA1.

5 Hybridize sample to flowcell30 Cluster GenerationBridge Amplification2. Amplify sample31 Cluster GenerationBridge Amplification2. Amplify sample32 : Cluster GenerationBridge Amplification2. Amplify sample33 Cluster GenerationBridge Amplification2. Amplify sample34dsDNA LinearizationCluster Generation3. Linearize LinearizationCluster Generation3. Linearize fragments36 Sequencing primerPrimer HybridizationCluster Generation4. Hybridize sequencing primer 37 MiSeqSequencing WorkflowData AnalysisSequencingCluster GenerationLibrary Preparation38 SequencingMiSeq Sequencing By Synthesis (SBS) DNA :Reversible Terminator Chemistry 1 4 ( ) OPPPHNNOO cleavagesitefluor3 blockODNAHNNOO3 O5 free 3 endXOH SBS Block Fluor 39 SBS TACG ddNTPs DNA G 3 TC.

6 GGGATGCTACGGGACYCLE 1 CYCLE 2 CYCLE 3 CYCLE 4 CYCLE 5 CYCLE 6 CYCLE 7 CYCLE 8 CYCLE 9 BASE CALLINGS ample PreparationCluster GenerationSequencing40 Add 4 Fl-NTP s + PolymeraseIncorporated FI-NTP imagedTerminator & fluorescent dye cleaved from FI-NTPX 36 -301 Sequencing by Synthesis (SBS) 41123789456T TTTTTTGT ..TGCTACGAT .. 1 Sequencing42 & 123 Read 2 Resynthesis & 43 Sequenced strand Index DNA Index Index Sequencing44 Sequenced strand DNA Index Sequencing45 Sequenced strand Index Index Sequencing PrimerIndex Sequencing46 Paired End Sequencing47 Sequenced strand 3 Sequenced strandBlocked 3 -endsPaired End Sequencing48 Bridge formation3 extension 3 Paired End Sequencing49 Double stranded DNAP aired End Sequencing 3 DNA DNA 50 Original forward strandBlocked 3 -ends ( )

7 Paired End Sequencing51 Reverse strandtemplateBlocked 3 -endsSequencing primer DNA 3 Read2 Read1 Paired End Sequencing52 Dual Index 53 Dual Index (i5 i7 2 123 Paired End Turnaround4 4 54 Region complementary to P5 grafting primer Index 2P5 primerDNA insertP7 primerIndex 1P7 grafting primerP5 grafting primerFlow cell surfaceDual Index Sequencing55 SBS Sequencing Primer Hybridization1. Read 1 Dual Index Sequencing56 Sequence (Cycle 1) 1 1 Dual Index Sequencing57 Sequence (Cycle 1) 1 1 Dual Index Sequencing58 Index 1 SeqPrimer 1 1 1 Dual Index Sequencing59 Index 1 read 8 1 1 1 1 Dual Index 1 1 1 1 Dual Index Sequencing61P5 grafting 1 1 1 1 2 Dual Index Sequencing627 dark cyclesP5 grafting 1 1 1 1 2 Dual Index Sequencing637 dark cyclesIndex 2 index read8 cyclesP5 grafting 1 1 1 1 2 2 Dual Index Sequencing647 dark cyclesIndex 2 index read8 cyclesP5 grafting 1 1 1 1 2 2 2 Dual Index Sequencing65 Original strandNew 1 1 1 1 2 2 2 Dual Index Sequencing66 New strandCleavage of Original 1 1 1 1 2 2)

8 2 Dual Index Sequencing67 Read 2 primer 1 1 1 1 2 2 2 2 Dual Index 1 1 1 1 2 2 2 2 2 Dual Index Sequencing69 Technology Spotlight Pub. No. 770-2013-J001 20 MAR2013 Spotlight 2 SBS Pub. No. 770-2013-J054 10 APR2014 702. 1 71 1 NGS 2 NGS 1 NGS 72 2 NGS 1. 2. 1 NGS 73 / / Q 74 / 75 FKBP8 Gene ExpressionRPS3 Gene ExpressionBrainUHRR3,115 reads31,109 reads 76 77 DNA DNA 2 kb DNA ( )78 de novo 79 DNA Repeat sequences Rearrangements De novo assembly Di-tag sequence cDNAs, ChIP, etc.

9 BAC-end sequencingrightwrong ( ) ( )Paired End Sequencing80 x = 81 x x 2 = 82 miseq MiSeqReagenetKit v3150600 Gb15 Gb 75bp x 2300bp x 2 25 M MiSeqReagenetKit v250300500 50bp x1150bp x2250bp x2 15 M 50 300bp 15M 25M83 25bp50bp100bp150bp250bp300bp10kb(Synthet ic) Small RNAChIP-SeqmRNAE xomeWhole GenomeDe novo assembly16S MetagenomicsLonger PCR products 84 This is really the best way to do sequencingThis is really the best way to do sequencingThis is really the best way to do sequencingThis isreally the best way to do sequencingThis is reallythe best way to do sequencingThis is really the best way to dosequencingThis is really the best way to dosequencing(----100 characters-------)

10 Skip OverviewPaired End Sequencing85 Read depth Fold coverage Coverage uniformity 86 Whole genome sequencingTruSeqPCR Free 30xWhole genome sequencingNexteraXT 40xdenovo AssemblyNexteraMatePair 60x Whole genome sequencingTruSeqPCR Free 30xExome SequencingNexteraRapid Capture 100xAmplicon SequencingTruSeqCustom Amplicon 30xAmplicon SequencingTruSeqCustom Amplicon ( 5%)500 Coverage uniformity depth Coverage uniformity 88 Human = Capture = 37 MbMiSeq150bp x2 = x2 = 120 GbHiSeq100bp x2 = 50Gb/lane 1 miseq 150bp x2 50 bp bp x = 4,500,000,000= 19 4,600,000 x 50 89 / / Q 90Q Quality Score = Phredscale Q Q30 1 Q Q30 91 NGS An Introduction to Next-Generation Sequencing Next-Generation Sequencing