Jun 27, 2018 - Pacbiosciecne (SMRT). First Generation. Second Generation. Third Generation*. *Some authors consider Nanopore as fourth generation.
Next Generation Sequencing On the Ascent
What is DNA sequencing: different generations of sequencing, why and what we need to know?
Aditya Arya, PhD Pathfinder Research and Training Foundation New Delhi
27th June 2018
How all this Started ? Nobel Laureate
Fred Sanger First Popular method of DNA Sequencing was pioneered
Cited by 72893 As on 27th June 2018 Image Credit: PNAS
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
Automation and Human Genome Project 1993 - 2001
Human Genome Project Clone by clone Approach (Consortium) Shotgun approach (Celera biotech)
Impact was huge !
Relied on Automated Sanger Sequencing
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
Beyond Human Genome Project Success of HGP driven many more initiatives Genomes available in NCBI :
37738 (as on 27
June 2018) Eukaryotes (6134); Prokaryotes (146790); Viruses (17630); Plasmids (12990); Organelles (11731) th
1000 Genome Project Cancer Genome Atlas ENCODE (Related encyclopedia of DNA elements) 100K Pathogen Genome Project … More to come 2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
The Quest begun …..
Faster Cost of HGP – Approx. 3 bn USD
Despite competition – 13 years
Cheaper 10+ countries , 1000+ researches involved
Entire life of your favorite dog
We can buy about 25 Boing - Dreamliner
Simpler Image credit https://www.newstrategygroup.com, Boing,
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
The Next Generation (Second Generation)
The need was already realised in early days And research begun in 1998 (Solexa, 454) But, first Sequencer was not commercialised Untill 2005!
NGS is faster , cheaper and simpler Image credit : Arya et al, Drawing Pin Publishing. 2018
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
What can be done using NGS Transcriptomics, Quantitative descriptive Alternate splicing , snRNA sequencing
Validation of genomic data, Reconstruction of maps and error finding.
Large scale discovery of Polymorphisms (SNPs and Copy number variations (CNVs)
Our focus In this workshop
Microbial identification Phylogenetic analysis Marine biotechnology Environmental biotech. Transcriptomics, Quantitative descriptive Alternate splicing , snRNA sequencing
Genome wide-Protein- DNA interactions , Active transcription factors, Binding sites etc.
Genome wide-Protein- DNA interactions , Active transcription factors, Binding sites etc.
Nucleosome positioning Image credit : Arya et al, © Drawing Pin Publishing. 2018
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
How to classify NGS? Initially There were three primary types
454 pyroseqeuncing Later it was sold to Roche
Solexa (genome analyser) Later it was sold to Illumina
ABI SoliD
Later it was sold to Life Technologies
Now when there are a large Number of players in NGS Its not early to classify them.
Basis for classification • • • • • •
Type of Technology Chemistry involved Detecton system Clonal amplification Manufacturers Generation
First Generation
Second Generation
Third Generation*
Pyroseqeuncing Sanger automated Sequencing
Sequencing by synthesis Illumina, thermo platforms
Nanopore technology Pacbiosciecne (SMRT)
*Some authors consider Nanopore as fourth generation.
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
How to Classify NGS Three things define NGS types Chemistry Involved : Ligation (SBL) | Synthesis
(SBS)
| Nanopore
Detection : Optical | Solid State | Electrical Amplication
: Clonal (Illumina, Ion torrent) | Single Molecule (Nanopore, Pacific)
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
Chemistries involved
Image credit: Mardis 2013, Goodwin, 2016
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
Detection System involved
Image credit: Mardis 2013, Goodwin, 2016
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
Clonal Requirements
Image credit – emulsion PCR binf.snipcademy.com
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
More on Chemistries … Sequencing by Ligation (SoLiD)
ABI SOLiD Image credit – Mardis, 2008; Goodwin et al, Annual Reviews.
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
More on Chemistries .. Sequencing by Ligation (cPAL) Combinatorial probe anchor ligation
Complete Genomics Image credit – Goodwin et al, Annual Reviews.
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
More on Chemistries .. Sequencing by Synthesis: CRT (Cyclic reversible Termination)
Detection system: (Optical) : Total Internal Reflection Fluorescence (TIRF) tris(2-Carboxymethylphosphine) or TCEP as cleaving agent Illumina Quiagen Image credit – Goodwin et al, Annual Reviews.
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
More on Chemistries .. Sequencing by Synthesis: SNA
(Single Nucleotide Amplification)
Detection system: (Optical) : charge coupled devices
454/Roche Pyrosequencer Image credit – Goodwin et al, Annual Reviews.
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
More on Chemistries .. Sequencing by Synthesis: SNA
Non – optical detecting system Detection system (solid state) Integrated complementary metal oxide semiconductor (CMOS) Ion sensitive field effect transistor (ISFET)
Image credit – Mardis, 2013l, Annual Reviews.
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Thermo – Ion Torrent Dr. Aditya Arya
More on Detection systems .. Optical detection Charge coupled devices (CCD)
Zero mode waveguides (ZMW)
Ion torrent platforms (Thermo, BGI)
Most Next generation platforms based on Luminescnce/ fluorescence (Illumina, Applied Biosciences etc.)
Image credit – Reuter 2015, Cell Press.
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
More on Detection systems .. Solid state and Electrical detection Solid state sensors
Ion torrent platforms (Thermo, BGI)
Electronic detection
Nanopore platforms (Oxford)
complementary metal-oxide semiconductor (CMOS) and an ion-sensitive field-effect transistor (ISFET) device Image credit – Reuter 2015, Cell Press.
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
Third Generation Sequencing SMRT Technology
SMRT : Single Molecule Real Time With 16 SMRTs (150,000 ZMW per SMRT bell) Read length ~ 40,000 nt
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
Third Generation Sequencing Nanopore Technology
Oxford nanopore
Different types of nanopores
Alpha hemolysin (1.5 x 5.2 nm)
Al2O3 (sub nm)
Msp 1 (1.2 x 3.7 nm) Graphene (sub nm)
Phi 29 (3.6 x 6 nm)
Al2O3, SiO3 (sub nm) Image credit –Feng et alr 2015,
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Image credit – Goodwin et al, Annual Reviews.
Dr. Aditya Arya
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
Glimpse of Platforms
Patterned flow cell
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
Glimpse of platforms
Oldest
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Latest
Dr. Aditya Arya
A comparative account
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
Basic Steps Adapter ligation and Library Preparation
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
Basic steps Library Amplification Emulsion PCR
Bridge amplification
2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
Basic steps
Next few speakers 2nd Introductory Course on Next generation Amplicon Sequencing. Chennai, India
Dr. Aditya Arya
Thank you and
happy learning
