454 Sequencing

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1 Roche Next Generation Sequencing: Making a Better Life GS FLX 454 lei.du@roche.com Lei Du, PhD, Director of Informatics 454 Life Sciences, Roche Applied Science

2 DNA?,, 细, 细遗传诊断 Genetic research, Disease mechanism Gene assisted breeding Biofuel development Plant/Animal evolution Infectious agent rapid monitoring Drug resistant mutations Cancer mutation, expression HLA typing Molecular diagnostics

3 DNA 测历 Late 90 s 454 Sequencing s 80 s Early 90 s Maxam-Gilbert 降 Sanger Next Generation 测

4 454 Sequencing Received Numerous Awards 年年创 2006 January 2007 Cover of Nucleic Acids Research

5 What can You use 454 Sequencing For? Turning Sequencing into Publications FAST, Straight Forward De Novo Sequencing Microorganisms (genome plasticity) Complete Plants, Animals BACs, YACs, Fosmids, Viruses Resequencing Whole Genome (e.g. the human genome) Disease associated regions Somatic mutations (cancer research via amplicon sequencing) Structural variations of the human genome 肿组 Transcriptome Analysis Expression profiling (e.g. SAGE-like, CAGE-like, GIS-PET EST-sequencing Full length cdna sequencing Gene Regulation Studies Identification of transcription factor binding sites (ChIP- Sequencing) Identification and quantification of sncrnas sequences Epigenetic Changes DNA-Methylation patterns DNA Metagenomes & Microbial Diversity Shotgun sequencing of the metagenome 16S amplicon sequencing 组 Ancient DNA Neanderthals, Mammoths and many more 400

6 Latest Research News Avian flu seqeuncing and assembly HBV resistance mutation SNP association: IFIH1 protecting against Diabetes Cucumber sequening and assembly (350 Mb) Oil Palm genome sequencing and assembly (1.8 Gb) 流糖尿组组

2008 Achievements Launched Titanium ~ 500 Mb/run Mode > 500 bp Q20 ~ 400 bp 10 hour run 190+ peer reviewed papers published in 2008 empowered by 454 Sequencing (335+ total) Ultra Deep Sequencing

7 2008 Achievements Launched Titanium ~ 500 Mb/run Mode > 500 bp Q20 ~ 400 bp 10 hour run 190+ peer reviewed papers published in 2008 empowered by 454 Sequencing (335+ total) Ultra Deep Sequencing Taking Off Mike Kozal: HIV Sequencing John Todd: Pools of amplicons Henry Erlich: HLA Sequencing Andy Fire: IgH and TcR sequencing Base Posn (%) 5% 4% 3% 2% 1% 0% Base Posn (bp) Q %

Selection A A B B Fragmentation Ligation Circularization A B

8 Titanium Paired End Processing 测组杂梁 Sample DNA 3, 8, or 20kb fragments Fragmentation A/B Ligation Amplification Size Selection A A B B Fragmentation Ligation Circularization A B ~150 bp ~150 bp A A B B 20 kb Gel Excision 8 kb Gel Excision See Poster 59

De novo Assembly: One library One Scaffold for Microbes S. pneumoniae 2.16 Megabases Scaffolds 1 (one) Contigs 2.10 Megabases N50 = 26,118 bases 链 Large Contigs = 155 E. coli 4.

9 De novo Assembly: One library One Scaffold for Microbes S. pneumoniae 2.16 Megabases Scaffolds 1 (one) Contigs 2.10 Megabases N50 = 26,118 bases 链 Large Contigs = 155 E. coli 4.64Megabases Scaffolds 1 (one) Contigs 4.54 Megabases 肠 N50 = 57,112 bases Large Contigs = 180 T. thermophilus HB Megabases Scaffolds 2 (two) Contigs 2.08 Megabases N50 = 10,469bases 热热 Large Contigs = 346 C. jejuni 1.64 Megabases Scaffolds 1 (one) Contigs 1.62 Megabases N50 = 153,789 bases 肠弯 Large Contigs = 29 组轻举 S. pneumoniae 8kb 2 E. coli 8kb 3 T. thermophilus 8kb 4 C. jejuni 8kb

10 Larger Genome Assembly Many other genomes in progress (insect, fish, fruit)

11 Plant Genomes de novo Sequencing Pinot Noir (500 MB) (WGS: 454, Sanger) Apple (750MB) (WGS: 454& Sanger) Melon (500 MB) WGS: (454, combo) Strawberry (200 MB) WGS: (Sanger, 454) Tomato (950 MB) (BACs) Potato (840 Mb) (BACs) Barley (5.4Gb) (BACs)

12 Sheep Skim Sequencing / SNP Discovery Courtesy John McEwan International Sheep Genomics Consortia (

24.5 Billion bases of genomic DNA sequencegenerated at 454 Sequencing Center and analysis performed by The Baylor Human Genome Center (Richard Gibbs and coworkers) 3.6 M variants detected 2.

13 24.5 Billion bases of genomic DNA sequencegenerated at 454 Sequencing Center and analysis performed by The Baylor Human Genome Center (Richard Gibbs and coworkers) 3.6 M variants detected 2.7 M in dbsnp; 0.9 M novel 6500 nssnps found in 4403 genes Identification of several disease susceptibility gene associations. Long reads and low bias give a more complete picture at a significantly lower oversampling rate than short read technologies DNA 现 James Watson

14 Amplicon and Ultra Deep Sequencing 454 测 Target specific sequence (ca. 25 bp) 454 A-primer (19 bp) 454 B-primer (19 bp) bp Locus specific PCR amplification Sequence of interest A B empcr & Sequencing >400bp, Library prep = simple PCR, HIV, &cdna

15 GS Amplicon Variant Analyzer Software Variation plot Variation from ref seq Reference sequence % variation # of reads Multiple alignment of reads against reference Variation key

Metagenomics and microbial diversity How it works

directly recovered from an environmental sample

anywhere soil, ocean water, tissue, mine shaft,

Metagenomic studies can give insight into:

Abundance of microbial species Gene content in a

Metatranscriptomics: analysis of the expressed

16 Metagenomics and microbial diversity How it works Metagenomics is the study of genetic material directly recovered from an environmental sample The environmental sample can be anything and from anywhere soil, ocean water, tissue, mine shaft, bee colony, gut, waste water, ulcers, air Metagenomic studies can give insight into: Microbial diversity in an environmental habitat Abundance of microbial species Gene content in a sample and the discovery of novel genes Metatranscriptomics: analysis of the expressed genome in a sample Viral pathogen detection Signature profile of an environment 组测环

17 Sequencing Infectious Agents 传检测

Influenza A virus subtype H5N1 流 Strains: A/Cygnus olor/germany/r1372/07 and A/Peking duck/germany/r1959/07) Reference Virus: A/swan/Germany/R65/2006, A/stone marten/germany/r747/2006.

18 Influenza A virus subtype H5N1 流 Strains: A/Cygnus olor/germany/r1372/07 and A/Peking duck/germany/r1959/07) Reference Virus: A/swan/Germany/R65/2006, A/stone marten/germany/r747/2006. Sanger sequence available. Total RNA was isolated from allantoic fluid (140 μl) using the QIAamp Viral RNA Mini Kit ( 尿 ) DNA syhnthesis using RT-PCR: SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity 11 segment specific PCRs: large segments 1, 2, and 3 were amplified in 2 fragments each empcr and 454 sequencing

19 11 Overlapping PCR Amplicons

20 Influenza H5N1 Assembly 1.45 Mb, 67x 0.76 Mb, 45x 1.59 Mb 0.57 Mb 40 MID 8 1 Titanium Can Sequence 300 Avian flu samples with multiplex barcoding scheme

21 Palacios et al. NEJM, 6 Feb 2008

22 454 Press conference Sunday April 22 nd, 2007

23 Clinical Course (culture, PCR, microarray) GS 100k DNA 14 LCMV (Lymphocytic Choriomeningitis Virus ) PCR ( ) ID# Age Diagnosis Organ Transplant Date Course 1 64 cirrhosis hepatitis C liver 12/04/06 fever, encephalopathy died day polycystic disease kidney 12/04/06 fever, CNS, graft rejection died day polycystic disease kidney 12/04/06 fever, CNS, graft rejection died day 29

Discovery of a pathogenic virus in humans 须测来较 DNA 列 Results: Clever sequence comparison to LCMV identifies virus as novel arenavirus: poor nucleotide match: longest continuous match is only 14

24 Discovery of a pathogenic virus in humans 须测来较 DNA 列 Results: Clever sequence comparison to LCMV identifies virus as novel arenavirus: poor nucleotide match: longest continuous match is only 14 nucleotides Query: LCMV: Query: LCMV: Query: LCMV: translation excellent amino acid match: Arenavirus and LCMV are closely related Read length is crucial in providing sufficient amino acid sequence information for correct identification of arenavirus as new viral species

25 HIV 测转录

26 Results 454

27 HBV

28 Disease Sequencing 肿, 遗传, 组测

29 Clonal Integration of a Polyomavirus in Human Merkel Cell Carcinoma Feng et al. Science January, 2008 T 酪 Merkel Cell Polyomavirus 细 (MCV) 8/10 Merkel Cell Carcinoma (MCC) MCV MCV MCC

30 Sugarbacker et al. March ( 4 1 ) 1 cdna 250 million base pairs were generated per sample. ~15,000 different gene entries were identified per sample (~3000 variants) ( 7, 3, 4 epigenetic silencing, 1 RNA editing) (49 3/7 ). XRCC6, PDZK1IP1, ACTR1A & AVEN.

易列测 Parallel resequencing of 10 cancer genes (177 exons) demonstrated an average 93% coverage over 7 cancer cell line and one normal genomic DNA samples.

31 易列测 Parallel resequencing of 10 cancer genes (177 exons) demonstrated an average 93% coverage over 7 cancer cell line and one normal genomic DNA samples. Multiple mutations and polymorphisms among 10 cancer genes were identified. Mutations and polymorphisms in the p53 gene were confirmed by traditional sequencing.

33 GS FLX is an enabling technology that is currently sold for Life Science Research Use Only GS,

目录 contents 09 th 封面 cover 华大 16 年感谢有你们 BGI 主办公共传播部总编杨碧澄文编南亚宁李茂娅李桂瑜美编刘宇璇公共传播部邮箱 bgibranding@genomics.cn 深圳华大基因研究院深圳市盐田区北山工业区综合楼 518083 深圳市

目录 contents 09 th 封面 cover 华大 16 年感谢有你们 BGI 主办公共传播部总编杨碧澄文编南亚宁李茂娅李桂瑜美编刘宇璇公共传播部邮箱 bgibranding@genomics.cn 深圳华大基因研究院深圳市盐田区北山工业区综合楼 518083 深圳市 WWW.GENOMICS.CN 21 SEP.2015 第 37 期总第 80 期以行践言,ICG 和你走过这 10 年 ICG10: 用行动为女性科学家加油对话业界大牛陈铮鸣 : 大队列与精准化医疗华大香港高通量测序实验室获得 CAP 认证天津华大成功搭建 Pacbio 平台