1. 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

2. 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

3. Sanger Sequencing

4. Microarray-High Throughput Assay

5. 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
= 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

6. 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

7. 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

8. Library Amplification (or not) DNA fragments (shotgun)
NGS Workflow Concept 1 4
DNA
RNA
Library Amplification (or not)
cDNA 5 2
Library
construction
DNA fragments (shotgun)
Sequencing 3 6
Add adaptor
Sequence Analysis
Without cloning

9. Single End (SE) / Pair End (PE)
NGS Workflow Concept
Read length
400 bp
200 bp
150 bp
25 bp
Single End (SE) / Pair End (PE)
2 X 150 bp SE PE
Total output = read length ╳ read numbers
-Total output 10 Gb, 100 bp read length:
-SE read numbers= 10 Gb / 100 bp= 100 M reads
-PE read numbers= 10 Gb / 200 bp= 50 M reads
Coverage = total output/sample size
-Total output 10 Gb, sample DNA size 1 Gb
-Coverage = 10 Gb/1 Gb = 10x
*Sample size normally means genome or transcriptome size.

10. Read length and sequence coverage
Sequencing depth
Low
High
Long read
high coverage
Coverage:
1.Genome de novo :100x-500x
2.Genome re-sequencing:30x-50x
3.RNA sequencing:>30x
4.RNA sequencing quantification:10x
Short read
low coverage
Long read
low coverage A B
Read length and sequence coverage
Example:
- Human transcriptome size: 30 Mb
1.Require RNA-seq output = 30 Mb * 30xCoverage=900 Mb
2.Require RNA-seq quantification output=30 Mb * 10xCoverage=300 Mb

11. Library Amplification
Platform-illumina
Library construction
Flow cell
Library Amplification
Bridge PCR
High resolution CCD

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

13. Platform-illumina family

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

15. Platform-Ion torrent
Ion PGM
Ion Proton

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

17. 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.

18. Multiplex Samples Sequencing-barcode
illumina
Ion torrent
PacBio

19. Future Platform-Nanopore

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

21. 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

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

23. RNA-seq Applications

24. 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.

25. 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: ~ nt
Molecular Cell 38, 2010

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

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

30. Year 2012 Mutations in KCND3 cause spinocerebellar ataxia type 22

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

33. Drug and Clinical Research

34. 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

35. EGFR inhibitor drug

36. Antibody Drug Conjugate (ADC)

37. 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

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

39. hsa-miR-122
hsa-miR-375

40. Non-Invasive Prenatal Testing (NIPT)

41. 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

42. Genome de novo Sequencing
BMC Genomics Nov 21;13:648

44. 牛樟芝-子實體和菌絲體為何效果不同?

45. 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.

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

47. 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?

48. Pathway analysis of
Metatranscriptome
16s rRNA change

49. NGS platforms videos 1.illumina: 2.Ion torrent: 3.PacBio RS:
2.Ion torrent:
3.PacBio RS:
4.Oxford Nanopore:
5.SOLiD:
6.Roche 454:

50. Thanks