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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
2003
1953
1985
1987
1977
1997
1990
DNA X-ray
structure
2005
2006
2011 2013
454
Sanger DNA
Sequencing
PCR
Microarray
illumina
Hiseq X
illumina
Solid
ABI370
sequencer
Human genome
project
2012
Human genome
project end
PacBio
Nanopore
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
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
Cost / per human genome
• High throughput sequencing in short time.
• Sequencing cost down than traditional
methods.
• High resolutions (~1 bp) and low noise.
Roche 454 GS
illumina Hiseq X :$1,000 /4 days
Clustering by bridge PCR
Fluorescence dye + dNTP
Library construction
Library Amplification
Sequencing
Semiconductor PH sensor
Ion PGM
Ion Proton
Loop adaptor
Single Molecule Real Time (SMRT)
-No Library PCR amplification
SMRT cell
ZMW: zero-mode waveguide
• 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
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
3.Epigenetic
4.Metagenomics
ChIP sequencing
DNA methylation sequencing
RNA
Coding
mRNA
Non-Coding
lncRNA
antisense
SnRNA
RNAi
rRNA
snoRNA
miRNA
siRNA
piRNA
tRNA
rasiRNA
scaRNA
RNA-seq Applications
Large 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
KCND3
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
=Effect type
=No effect type
Bad Results
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
Metagenome/Metatranscriptome
Metagenome
Metatranscriptome
Microbial DNA
Microbial RNA
16s rRNA-seq
Who is there?
Shotgun whole
genome-seq
Who is there?
What they have?
RNA-seq
How are they doing?
16s rRNA change
Pathway analysis of
Metatranscriptome
NGS platforms videos
1.illumina:
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