1. NGS technologies

2. First generation
Maxam-Gilbert
Sanger

3. Sanger sequencing

4. Automatization: first generation
Possible due to:
the replacement of phosphor tritrium-radiolabelling with fluorometric based detection (allowing the reaction to occur in one vessel instead of four)
improved detection through capillary based electrophoresis.
Read length: slightly less than 1kb
Shotgun sequencing: Clone overlapping fragments and sequence.
Enhancements:
PCR (in vitro cloning)
Recombinant DNA technologies
More efficient DNA polymerases
Newer sequencers allowed for simultaneous sequencing of multiple samples

5. The second generation: pyrophosphate synthesis
Advantages:
Natural nucleotides were used (instead of heavily modified dNTPs)
Bases could be read real-time, without requiring electrophoresis
Enhancements:
Attaching DNA to paramagnetic beads
Removing unincorporated dNTPs enzymatically (without the need to wash)
Challenge:
Determine the number of attached nucleotides in a row (the GG and CC cases)

6. 454 Roche
Pyrosequencing was later licensed to 454 Life Sciences, a biotechnology company founded by Jonathan Rothburg, where it evolved into the first major successful commercial ‘next-generation sequencing’ (NGS) technology. Later purchased by Roche.
Paradigm shift:
mass parallelisation of sequencing reactions due to microfabrication and high-resolution imaging

7. DNA nebulization => fragments of length 50-900 bp
Fragments are blunt-ended and phosphorylated with polymerases (polishing reaction)
Adapter ligation: 44-base adaptors – containing in 5’-3’ direction:
20b PCR primer
20b sequencing primer
4b AGTC sequencing key
Two classes of adaptors: A and B, differing in sequence and in presence of 5’ biotin tag in B adaptor
Size selection: gel electrophoresis followed by cutting the regions for bp
Single stranded AB-adapted library prepared
Attachment to beads, emulsion PCR
Double stranded amplified DNA melted to single strands
Enrichment primer added to filter against null beads
Sequencing primer added
Incubation with DNA polymerase, SSB protein, apyrase – on fibreoptic slide
Luciferase preparation
Signal normalization (account for the number of beads per well, and the number of template sequences per bead):
(i) raw signals are first normalized by reference to the pre- and post-sequencing run PPi standard flows, (ii) these signals are further normalized by reference to the signals measured during incorporation of the first three bases of the known “key” sequence included in each template.

8. The Solexa-Illumina system
Shankar Balasubramanian,
David Klenerman
very early experiments included the observation of single molecules of DNA polymerase binding to substrate DNA (template plus primer) in solution, using a highly sensitive confocal single molecule
detection system
(cleavable)
dye-labaled dNTPs

9. 1997: Solexa sequencing concept
Basic => applied science transition
Chemical blockage of dNTPs
Mutagenesis of DNA polymerase

10. 1997: Solexa sequencing concept
Basic => applied science transition
2. Solid phase sequencing
Goal:
Theoretical limit was estimated to be one sequenceable DNA
fragment per diffraction limited site (less than 1 square mm) =>
Aim was to sequence one billion bases (~ human genome size)
Fragmentation drive parallelization was possible with consideration of future alignment of the reads to a ‘master’ genome.

11. Advancements to solexa sequencing concept: solid phase DNA amplification
a stronger signal,
a less expensive imaging system
reduction of stochastic single molecule errors

12. An image of the surface taken during a cycle of an early Solexa sequencing experiment.
Each of the spots is a cluster of identical DNA sample fragments and the colour indicates which of the four bases has been incorporated at that particular cycle.

13. Advancements to solexa sequencing concept: paired-end sequencing
sequencing of one end of the original DNA fragment, followed by the other end to facilitate de novo sequencing of genomes

14. Timeline
2006, Solexa-Illumina Genome Analyzer, 1 bln bases, 1G of human genome (2.5 days to generate read lengths of 36 bases)
The first human African, Asian and cancer genomes plus the first giant panda
genome were sequenced on the Genome Analyser.
2010, Illumina HiSeq 2000, 200 bln bases, 200G of genome (2 human genomes with x30 coverage)
Five-fold increase in sequencing capacity annually (more than 2-fold of the Moore’s law)

15. Illumina sequencing technologies
Details
Illumina sequencing technologies

16. General workflow

17. Fragmentation
Genome Analyzer was done by nebulisation (in 30-60% glycerol at 30-35psi)
fragments were of range bp and a peak around 5-600bp
reproducible and cheap technique
But uneconomical:
200bp +/- 20bp fragments represent only ∼10% of the total DNA
half of the DNA vaporises during nebulisation
Thus only 5% of the original DNA is used for subsequent library generation.
Acoustic energy is controllably focused into the aqueous DNA sample, resulting in cavitation events. The collapse of bubbles in the suspension creates multiple, intense, localized jets of water, which disrupt the DNA molecules.
Range = bp
200bp fragments comprise 17% of the total fractionated DNA
very little DNA is lost
Narrow size distribution allows to skip the size-selection step in some applications

18. Size selection 1. Size detection 2. Slicing
3. Gel melting and DNA extraction: A/T bias

19. General workflow

20. Fragment processing, adapter ligation
Illumina:
Fragments are blunted and 5’ phosphorylated with enzymes (polymerases and kinases)
the 3′ ends are A-tailed
Adapters are ligated with T-overhangs (sticky end ligation)
Adapters can be barcoded for different samples
Ion torrent:
Fragments are blunted
Adapters are blunt-end ligated

21. Fragment processing, adapter ligation

22. General workflow

23. Size-selection, PCR amplification
Size selection is for
Filtering against adapter-dimers
Filtering against chimeric fragment ligations
PCR amplification
Increases depth or coverage
May lead to over-duplication (PCR duplicates)
High amount of initial template DNA may lead to single-stranded fragments

24. General workflow

25. Bridge amplification

26. General workflow

27. Sequencing by synthesis

28. Sequencing by synthesis

29. Paired-end sequencing: long (d) and short (c) inserts

30. Illumina machine comparison

31. Illumina advantages Advantages High throughput/ cost
Wide range of applications
Disadvantages
High substitution error rates
Sequence quality deterioration towards the end

34. Summary: SOLiD has one of the lowest error-rates (~0
Summary: SOLiD has one of the lowest error-rates (~0.01) due to 2-base encoding. It is however still limited by short read lengths ( 35 bp / 85 bp for PE).

40. Comparison of sequencing technologies

43. 454
Pros: long reads
Cons: low throughput, high reagent cost, high error rates in homopolymers
Illumina/Solexa
Pros: leader => most library prep protocols are for Illumina;
highest throughput and lowest cost per base;
Reads up to 300 bp
Cons: before hiseq X, random scattering of fragments across the flow cell made it concentration-dependent;
Requires high complexity of library (?)
SOLiD
Pros: highest throughput; lower error rates
Cons: short treads (75 nt); less widely used
Ion Torrent:
Pros: semi-conductor – no need for optical scanning and fluorescent nucleotides;
Fast run-times
Cons: high error-rates for homopolymers
PacBio
Pros: extremely long reads (up to 20 kb); fast run-times
Cons: high-cost; high error-rate; low throughput