1. Next Generation Sequencing
Rahul Krishnan
AAH.MA5.03

2. Introduction
Nucleic acid sequencing - method for determining the arrangement of nucleotides present in a given DNA or RNA molecule
1970s, Sanger and colleagues and Maxam and Gilbert developed - chain termination and fragmentation techniques
Incentive for developing new strategies :
Reductions in the cost
The potential utility of short-read sequencing
Development of new molecular methods
General progress in technology across disparate fields
Automated Sanger method -‘first-generation’ technology, & newer methods - next-generation sequencing (NGS).

3. Sanger sequencing
Cycles of template denaturation, primer annealing and primer extension - cycle sequencing
Primer extension is terminated by incorporation of fluorescently labeled dideoxynucleotides (ddNTPs)
The label on the terminating ddNTP of any fragment corresponds to the nucleotide identity of its terminal position
Sequence is determined by high-resolution electrophoretic separation in polymer gel and Laser excitation of fluorescent labels

4. First generation Technology
Third Generation
Third Generation
Next Generation Technology
Next Generation Technology

5. NGS technologies
The first NGS technology (2005 ) - the pyrosequencing method by 454 Life Sciences (now Roche)
2006- the Solexa/Illumina sequencing plat-form was commercialized (Illumina acquired Solexa in 2007).
In Sequencing by Oligo Ligation Detection (SOLiD) by Applied Biosys-tems (now Life Technologies)
In 2010, Ion Torrent (now Life Technologies) released the Personal Genome Machine (PGM).
PGM uses semiconductor technology and does not rely on the optical detection of incorporated nucleotides using fluorescence and camera scanning. This resulted in higher speed, lower cost, and smaller instrument size.

6. 454 Pyrosequencing

7. 454 pyrosequencing
Clonal sequencing features are generated by emulsion PCR, with amplicons captured to the surface of 28-μm beads
Beads are treated with denaturant to remove untethered strands & subjected to a hybridization-based enrichment for amplicon- bearing beads
A sequencing primer is hybridized to the universal adaptor at the appropriate position and orientation
The amplicon-bearing beads are preincubated with Bacillus stearothermophilus (Bst) polymerase and single-stranded binding protein
Deposited on to a microfabricated array of picoliterscale wells contining Smaller beads bearing immobilized enzymes - ATP sulfurylase and luciferase.
One side of the semi-ordered array - flow cell & the other side is bonded to a fiber-optic bundle for CCD (chargecoupled device)-based signal detection.

9. The Solexa/Illumina sequencing platform

10. The Solexa/Illumina sequencing platform
Libraries - mixture of adaptor-flanked fragments up to several hundred base-pairs (bp) in length. Amplified sequencing features are generated by bridge PCR
In this approach, both forward and reverse PCR primers are tethered to a solid substrate by a flexible linker,
There by amplicons arising from any single template molecule remain immobilized and clustered to a single physical location on an array.
Alternating cycles of extension with Bst polymerase and denaturation with formamide
The amplicons are single stranded (linearization) and a sequencing primer is hybridized to a universal sequence
Nucleotides are ‘reversible terminators’, - chemically cleavable moiety at the 3′ hydroxyl position allows only a single-base incorporation to occur in each cycle

12. Sequencing by Oligo Ligation Detection (SOLiD)

13. Sequencing by Oligo Ligation Detection (SOLiD)
Clonal sequencing features are generated by emulsion PCR, with amplicons captured to the surface of 1-μM paramagnetic beads
Beads bearing amplification products are immobilized to a solid planar substrate to generate a dense, disordered array
A universal primer complementary to adaptor sequence is hybridized to the array of amplicon-bearing beads
Sequencing by synthesis is driven by a DNA ligase with fluorescently labeled octamer
The octamer mixture is structured, in that the identity of specific position(s) within the octamer (e.g., base 5) correlate with the identity of the fluorescent label.
After ligation, images are acquired & octamer is chemically cleaved between positions 5 and 6
Upon completing several such cycles, the extended primer is denatured to reset the system

15. Ion Torrent sequencing

16. Ion Torrent sequencing
Combines computer software with integrated circuits and complementary metal-oxide semiconductors (CMOS).
Adopts an electrochemical detection system called ion-sensitive field-effect transistors (ISFET)
Detecting and analyzing the release of a hydrogen ion (or proton) on addition of a nucleotide triphosphate
The proton release causes a slight pH shift which is detected by a CMOS sensor
Each chip has at least 1.2 million sensors.
Fragments of DNA are ligated to adapters and clonally amplify adapter-ligated libraries onto acrylamide beads
DNA polymerase and primers are bound to templates and filled to loading port
DNA fragments and DNA polymerase are sequentially flooded with unmodified dNTPs
The hydrogen ion that is released in the reaction changes the pH of the solution, which is detected by an ISFET

18. Other NGS platforms Polony sequencing (Helicos BioSciences)
The template DNA is not amplified before sequencing, which places this method at the interface between NGS and the so-called third- generation sequencing technologies.
Pacific Biosciences (PacBio)
PacBio is based on the detection of natural DNA synthesis by a single DNA polymerase. Incorporation of phosphate-labeled nucleotides leads to base-specific fluorescence, which is detected in real time

19. Pac Bio single molecule, real-time sequencing technology (SMRT)

20. SMRT
Sequencing-by-synthesis technology based on real-time imaging of fluorescently tagged nucleotides
The sequencing reaction ends when the template and polymerase dissociate
Uses phosporyated nucleoties with fluorescent tag at phospate group
Utilizes a zero-mode waveguide (ZMW). A single DNA polymerase enzyme is affixed at the bottom of a ZMW with a single molecule of DNA as a template
Once nucleotide is incorporated - fluorescent tag is cleaved off and diffuses out of the observation area of the ZMW
Read lengths of 2.5 to 2.9 kilobases
Released on 2010 , upgraded versions on 2011,2013 , now PacBio RS II with 5-7 Kb read length

24. Applications of next-generation sequencing
Category
Examples of applications
Complete genome resequencing
Comprehensive polymorphism and mutation discovery in individual human genomes
Reduced representation sequencing
Large-scale polymorphism discovery
Targeted genomic resequencing
Targeted polymorphism and mutation discovery
Paired end sequencing
Discovery of inherited and acquired structural variation
Metagenomic sequencing
Discovery of infectious and commensal flora
Transcriptome sequencing
Quantification of gene expression and alternative splicing; transcript annotation; discovery of transcribed SNPs or somatic mutations
Small RNA sequencing
microRNA profiling
Sequencing of bisulfite-treated DNA
Determining patterns of cytosine methylation in genomic DNA
Chromatin immunoprecipitation– sequencing (ChIP-Seq)
Genome-wide mapping of protein-DNA interactions
Nuclease fragmentation and sequencing
Nucleosome positioning
Molecular barcoding
Multiplex sequencing of samples from multiple
individuals

25. Conclusion
NGS technologies have an impressive range of applications, and more are being developed
NGS technologies are being used to characterize the evolutionary relationships of ancient genomes and to elucidate the role of noncoding RNAs in health and disease
The field of NGS development and applications is a fast-moving area of research