Transcription of NGSをはじめよう！これだけは知っておきたいMiSeq 解析原 …
1 2012 Illumina, Inc. All rights reserved. Illumina, illuminaDx, BaseSpace, BeadArray, BeadXpress, cBot, CSPro, DASL, DesignStudio, Eco, GAIIx, Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, Infinium, iSelect, miseq , Nextera, Sentrix, SeqMonitor, Solexa, TruSeq, VeraCode, the pumpkin orange color, and the Genetic Energy streaming bases design are trademarks or registered trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners. NGS miseq June 27, 2014 2 miseq miseq miseq Reporter Outline 3 miseq NGS 1 ng~1 g DNA(RNA) DNA Sequencing By Synthesis (SBS 15 Gbases 2013 6 4 ChIP-Seq Small RNA QC HiSeq2500 1000 Gb / 6 / miseq v2 ~8 Gb / 2 (39 ) / miseq v3 ~15 Gb / 3 (55 ) / NextSeq 500 120 Gb / 1 / miseq 5 miseq Illumina Experiment Manager (IEM) miseq miseq Reporter 1.)
2 2. 3. 4. 6 miseq miseq 7 miseq miseq Control Software (MCS) 8 miseq 1. 2. 3. 4. Illumina Experiment Manager (IEM) miseq miseq Reporter 9 qPCR Bioanalyzer Flowcell Index ( DNA : TruSeq DNA HT (Dual Index) 10 ( ) 2013 Bioanalyzer Library QC 2013/10/18 11 DNA TruSeq DNA prep DNA (1 ug ) DNA bp 2 DNA 12 TruSeq Nano DNA TruSeq DNA PCR-Free Nextera XT Nextera Mate Pair TruSeq Custom Amplicon Nextera Rapid Capture Exsome Nextera Rapid Capture Custome Enrichment TruSight Myeloid TruSeq RNA TruSeq Stranded mRNA TruSeq Stranded Total RNA TruSeq targeted RNA Panel TruSeq Small RNA TruSeq ChIP TruSight Tumor Nextera DNA TruSeq Custom Amplicon Caner Panel TruSeq RNA Access Library Kit TruSight 2014 6 13 Kit Selector 2014/03/31 ( ) 14 miseq DNA Illumina Experiment Manager (IEM) 1.)
3 2. 3. 4. miseq miseq Reporter 15 Illumina Experiment Manager Illumina Experiment Manager (IEM) (MyIllmina ) 16 : (.csv) 17 18 Category Workflow 19 Targeted Resequencing TruSeq Amplicon PCR Amplicon Metagenomics 16S rRNA Enrichment Amplicon - DS Small Genome Sequencing Resequencing Plasmids Assembly RNA Sequencing Targeted RNA Small RNA RNA-Seq Other Library QC FASTQ Only ChIP-Seq 20 Run 21 Index Warning 22 : Valid 23 24 miseq miseq 1. 2. 3. 4. miseq Reporter Illumina Experiment Manager (IEM) 25 miseq miseq 26 miseq 27 miseq V2 50 cycle 50 bp 2x26+23 bp 15 M (Small RNA ) Library QC V2 300 cycle 300 bp 2x151+23 bp 15 M Nextera XT PCR TruSeq Custom Amplicon V2 500 cycle 500 bp 2x251+23 bp 15 M De novo miseq V3 - 150 cycle 150 bp 2x76+23 bp 25 M RNA-seq ( ) Exome V3 600 cycle 600 bp 2x301+23 bp 25 M de novo HLA miseq dual index 28 DNA DNA 1.
4 2. 4. 1. DNA 2. PCR 3. miseq 29 Sequencing By Synthesis (SBS) 3 DNA 1 4 O PPP HN N O O 3 O DNA HN N O O 3 O 5 X OH 1 SBS 30 SBS T A C G ddNTPs DNA G 3 T C .. G G G G A 31 Quality scores (Q Scores) Q scores base call Q Score Probability of error Q30 1 in 1000 Q20 1 in 100 1-4 = T G C T SBS 32 ( ) 2013 SBS (Sequencing By Synthesis) 2013/07/19 33 7 11 NGS Online Truobleshooter 7 25 miseq 8 miseq 34 miseq miseq Reporter 1.
5 2. 3. 4. miseq Illumina Experiment Manager (IEM) 35 miseq Reporter (MSR) miseq FASTQ miseq Reporter (MSR) miseq Control Software(MCS) /Real Time Analysis (RTA) SAV Windows PC Or 36 Alignments and Variant Detection Images/TIFF files Base Calling Intensities Outputs Outputs Analysis Type Primary Analysis (RTA) Secondary Analysis (MSR or BaseSpace) Sequencing (MCS) miseq 37 16S De novo Enrichment Targeted RNA Small RNA QC 16S de novo Small RNA QC Targeted RNA Enrichment miseq Reporter (MSR) 38 miseq Reporter 2013 miseq Reporter 2013/11/01 Reference Guide miseq Reporter Theory of Operation 39 Resequencing Enrichment Metagenomics : SNV Insertion / deletion duplicate % + miseq Reporter (MSR) 40 miseq miseq miseq Reporter Summary 41
