次世代基因體定序技術在生醫研究與生技產業之應用 國瑞有限公司 洪國勝博士

History of Gene Research Human genome project end NGS 2003 1985 2011 2013 1987 2005 1953 1977 1997 2006 2012 1990 454 DNA X-ray structure Sanger DNA Sequencing illumina Hiseq X PCR Microarray illumina Solid PacBio ABI370 sequencer Human genome project end Nanopore Human genome project Ion Torrent

Sanger Sequencing

Microarray-High Throughput Assay

Limits of Sanger Sequencing Preparation Library >Vector and Host problems. 1:3 / 2:1 ? Amp conc.? DH5a/XL1/JM109? >Time:? >Cost:? Sequencing Run time >ABI 3730xl: 1 hr / run, output 90,000 bp >Human = 3,000,000,000 bp / 90,000 bp*1hr =333333.33 hr = 1389 days = 3.8 years Sequencing Cost >NTD 180/1,000bp >Human=3,000,000,000 bp / 1,000 bp*NTD 180 =NTD 540,000,000 ABI 3730xl

Limits of Microarray Resolution: 50~100 bp - Unable to explore the differences between single bp. Need reference gene information: - Can not be used in unknown sequence species. Hybridize: -Easily lead to false positive. Relative quantification: -Not reflect the actual situation of gene expression. High background noise

Advantages of NGS High throughput sequencing in short time. Sequencing cost down than traditional methods. High resolutions (~1 bp) and low noise. Roche 454 GS Cost / per human genome illumina Hiseq X :$1,000 /4 days

Clustering by bridge PCR Platform-illumina Clustering by bridge PCR Fluorescence dye + dNTP

Platform-illumina family

Library Amplification Semiconductor PH sensor Platform-Ion torrent Library construction Library Amplification Emulsion (Em) PCR Semiconductor PH sensor Sequencing

Platform-Ion torrent Ion PGM Ion Proton

Platform-PacBio RS SMRT cell Single Molecule Real Time (SMRT) -No Library PCR amplification Loop adaptor SMRT cell ZMW: zero-mode waveguide

Platform-PacBio RS Extraordinarily long reads: Produce reads with average lengths of 4,200 to 8,500 bp, with the longest reads over 30,000 base pairs.

Future Platform-Nanopore

NGS Applications-Human Health Genome Exome Transcriptome DNA-protein interaction DNA modifications

DNA methylation sequencing NGS Applications 1.RNA-sequencing mRNA 2.RNA-quantification RNA 3.Small RNA sequencing miRNA/other small RNA 4.LncRNA sequencing Large non-coding RNA 5.Metatranscriptome Re-sequencing 1.Genome sequencing de novo sequencing 2.Exom sequencing DNA ChIP sequencing 3.Epigenetic DNA methylation sequencing 4.Metagenomics

RNA Coding Non-Coding SnRNA RNAi mRNA lncRNA antisense tRNA rRNA snoRNA scaRNA miRNA siRNA piRNA rasiRNA

RNA-seq Applications

Large non-coding RNAs Small non-coding RNAs lncRNA -Full name: Long intergenic non-coding RNAs -Size: >200 nt. Small non-coding RNAs siRNA -Full name: Small interfering RNA -Size: 20~25 bp. miRNA -Full name: microRNA -Size: ~21-24 nt. piRNA -Full name: Piwi-interacting RNA -RNA-Size: ~26-31 nt. rasiRNA -Full name: Repeat associated small interfering RNA -RNA-Size: ~24-29nt.

Non-Coding Nuclear RNA Small Nuclear RNAs (snRNAs) snoRNA -Full name: Small nucleolar RNAs -Size: 70~120 nt. scaRNA -Full name: Small Cajal body-specific RNA -Size: ~120-300 nt Molecular Cell 38, 2010

Exome-seq Cost-effective alternative to whole-genome sequencing

Year 2002 1p21-q23 75 MB region and >1000 genes…..

Year 2012 Mutations in KCND3 cause spinocerebellar ataxia type 22

Epigenetic Study The study of heritable changes in gene function that occur without a change in the DNA sequence. DNA methylation/Histone modification.

Drug and Clinical Research

Bad Results NGS genotyping Good Results =Effect type =No effect type New Drug A NGS genotyping New Drug A New Drug A Good Results New Drug B

EGFR inhibitor drug

Antibody Drug Conjugate (ADC)

Circulating Nucleic Acids Isolated from serum or plasma. They are increasingly recognized as biomarkers for cancers/hematological malignancies/fetal health monitor. DNA:<1000 bp RNA:<1000 nt miRNA:~20 nt

Circulating Nucleic Acids-Exome Sequencing Nature 497, 108–112 , 2013

hsa-miR-122 hsa-miR-375

Non-Invasive Prenatal Testing (NIPT)

Genome Sequencing de novo sequencing. Re-sequencing (mapping to reference sequence). Pathogenicity/Antimicrobial resistance study. Application in bio-resource development: Industrial Enzymes- Cellulase, Xylanase, Lipase…. Biomass Energy pathways- Ethanol, n-Butanol, Hydrogen.... Secondary metabolites synthases- Penicillin, Tetrodotoxin, Steroids… Assemble/ mapping to reference DNA fragments reads Genome sequence

牛樟芝-子實體和菌絲體為何效果不同?

Metagenome/Metatranscriptome Metagenomics/Metatranscriptoms is the study of all genomes/transcriptomes present in any given environmental. No identifications. Environment study: -Microbial population. Individual health: -Disease. -Immunity. -Metabolism.

NGS application in Metagenome/ Metatranscriptome 16s rRNA-seq Genome-seq RNA-seq Without cloning

Metagenome/Metatranscriptome Shotgun whole genome-seq Microbial DNA Microbial RNA 16s rRNA-seq Shotgun whole genome-seq RNA-seq Who is there? Who is there? How are they doing? What they have?

Pathway analysis of Metatranscriptome 16s rRNA change

NGS platforms videos 1.illumina: 2.Ion torrent: 3.PacBio RS: https://www.youtube.com/watch?v=l99aKKHcxC4 2.Ion torrent: https://www.youtube.com/watch?v=WYBzbxIfuKs 3.PacBio RS: https://www.youtube.com/watch?v=NHCJ8PtYCFc 4.Oxford Nanopore: https://www.youtube.com/watch?v=3UHw22hBpAk 5.SOLiD: https://www.youtube.com/watch?v=nlvyF8bFDwM 6.Roche 454: https://www.youtube.com/watch?v=bFNjxKHP8Jc

Thanks