1. Sequencing techniques
BS222 – Genome Science Lecture 2
Sequencing techniques
Vladimir Teif

2. Module structure
Genomes, sequencing projects and genomic databases (VT) (Oct 9, 2018)
Sequencing technologies (VT) (Oct 11, 2018)
Genome architecture I: protein coding genes (VT) (Oct 16, 2018)
Genome architecture II: transcription regulation (VT) (Oct 18, 2018)
Genome architecture III: 3D chromatin organisation (VT) (Oct 23, 2018)
Epigenetics overview (PVW) (Oct 25, 2018)
DNA methylation and other DNA modifications (VT) (Oct 30, 2018)
NGS applications I: Experiments and basic analysis (VT) (Nov 1, 2018)
NGS applications II: Data integration (VT) (Nov 8, 2018).
Comparative genomics (JP, guest lecture) (Nov 13, 2018)
SNPs, CNVs, population genomics (LS, guest lecture) (Nov 15, 2018)
Histone modifications (PVW) (Nov 20, 2018)
Non-coding RNAs (PVW) (Nov 22, 2018)
Genome Stability (PVW) ) (Nov 27, 2018)
Transcriptomics (PVW) (Nov 29, 2018)
Year's best paper (PVW) (Dec 6, 2018)
Revision lecture (all lecturers; spring term)

4. DNA polymerization reaction
Pyrophosphate

5. Polymerase chain reaction (PCR)
Developed in 1983 by Kary Mullis (Nobel Prize in 1993)
PCR usually employs a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the thermophilic bacterium Thermus aquaticus.

6. Real-time PCR (a.k.a. quantitative PCR, qPCR)
The strength of the fluorescent signal increases with increasing the amount of single-stranded DNA in solution at each time point of the PCR amplification

7. DNA sequencing
DNA sequencing is the process of determining the sequence of nucleotides (As, Ts, Cs, and Gs) in a piece of DNA. We use it for:
Gene sequencing:
predict structure and function of the product
predict functionally important parts of the sequence
compare sequence across species (orthologues)
compare members of a gene family (paralogues)
Genome sequencing:
find all genes and non-coding regulatory regions
study the structure and organization of the genome
Metagenomics:
enumerate microbes without culturing
(soils, sediments, gut microbiom)

8. 1st Generation Sequencing
Maxam–Gilbert sequencing (“chemical sequencing”)
Sanger sequencing (“chain termination sequencing”)

9. Maxam–Gilbert sequencing (1976)
Developed by Allan Maxam & Walter Gilbert in 1976–1977.
Based on nucleobase-specific partial chemical modification of DNA and subsequent cleavage of the DNA backbone at sites adjacent to the modified nucleotides.
Almost not used nowadays

10. Sanger sequencing (1977)

11. Sanger sequencing is based on the use of dideoxynucleotides
In a regular nucleotide dNTP, the 3’ hydroxyl group acts as a “hook," allowing a new nucleotide to be added to an existing chain.
In dideoxy nucleotide ddNTP there is no hydroxyl available and no further nucleotides can be added. The chain ends with the dideoxy nucleotide
Image credit: OpenStax College, Biology

12. Sanger sequencing is also called “chain termination” sequencing

13. Sanger sequencing: fluorescent labels

14. Sanger sequencing nowadays

15. Sanger sequencing uses & limitations
Sanger sequencing gives high-quality sequence for relatively long stretches of DNA (up to about 1000 bp)
It's typically used to sequence individual pieces of DNA, such as bacterial plasmids or DNA copied in PCR
Sanger sequencing is expensive and inefficient for larger-scale projects, such as the sequencing of a human genome
Sanger sequencing was used in the Human Genome Project ( ), but nowadays it is not used to sequence human genomes

16. The first human genome was sequenced with the shotgun method using Sanger sequencing technique
Idea: chop the genome into pieces, and sequence them

17. Hierarchical shotgun method

18. Next Generation Sequencing (NGS)
Parallelized (sequence millions DNA fragments simultaneously)
High-throughput (can sequence human genome in ~1 day)
Cost effective (~£1,000 per human genome)
Many competing methods:
Pyrosequencing (Roche 454; discontinued in 2013)
Sequencing by ligation (SOLiD; discontinued in 2016)
Sequencing by synthesis (Illumina; major market share)
IonTorrent/Ion Proton (ThermoFisher; limited market share)
Nanopore sequencing (Oxford Nanopore; on the rise) …

21. Illumina (Solexa) sequencing
Watch this video (4.30 min):
As you are watching it, you can type any questions on Padlet:
You can also reply to your peers’ questions `

22. Illumina (Solexa) sequencing
After PCR has been carried out to amplify each DNA fragment (“read”), many clusters with many copies of the same read have been created on the slide. They are then separated into single strands to be sequenced.
The chip is then flooded with nucleotides and DNA polymerase. These nucleotides are fluorescently labelled. They also have a terminator, so that only one base is added at a time.

23. Illumina (Solexa) sequencing
An image is taken of the slide. In each read location, there will be a fluorescent signal indicating the corresponding nucleotide that has been added.
The slide is then prepared for the next cycle. The terminators are removed, allowing the next base to be added, and the fluorescent signal is removed, preventing the signal from contaminating the next image.

24. Illumina (Solexa) sequencing
This process is repeated, adding one nucleotide at a time and imaging in between.

25. Illumina (Solexa) sequencing
Computer detects the base at each site on the slide in each image and these are used to reconstruct the DNA sequences

26. Roche 454 (“Pyrosequencing”)
Pyrosequencing relies on light detection based on a chain reaction when pyrophosphate is released.
DNA fragments are attached to beads, one DNA fragment per bead. The fragments are then amplified by PCR.
Each bead (covered in many PCR copies of a single sequence ) is placed in a single well of a microfabricated microarray.

27. Pyrosequencing chemistry

28. Roche 454 (“Pyrosequencing”)
The slide is flooded with one of the four nucleotides. Where this nucleotide is next in the sequence, it is added to the read.
The addition of each nucleotide releases a light signal. These locations of signals are detected and used to determine which beads the nucleotides are added to. 

29. Roche 454 (“Pyrosequencing”)
This nucleotide mix is washed away. The next nucleotide mix is then added and the process repeated, cycling through the four nucleotides.

30. Roche 454 (“Pyrosequencing”)
This generates graphs for each sequence read, showing the signal density for each nucleotide wash. The sequence can then be determined computationally from the signal density in each wash.

31. Ion Torrent/Ion Proton sequencing
Ion torrent and Ion proton sequencing exploit the fact that addition of a dNTP to a DNA polymer releases an H+ ion
 pH of the solution changes
Like 454, the slide is flooded with a single species of dNTP, along with buffers and polymerase, one NTP at a time. The pH is detected in each of the wells, as each H+ ion released will decrease the pH.
The changes in pH allow us to determine if that base, and how many thereof, was added to the sequence read.

32. Ion Torrent/Ion Proton sequencing
Nucleotides are washed away, and the process is repeated cycling through the four nucleotide species.

33. Ion Torrent/Ion Proton sequencing
The pH change, if any, is used to determine how many bases (if any) were added with each cycle for each analysed DNA fragment

34. Nanopore “strand sequencing”
Nanopore “strand sequencing” is a technique that passes intact DNA polymers through a protein nanopore, sequencing in real time as the DNA translocates the pore. The type of nucleotide passing the nanopore is detected electronically (not optically).
Up to ~1Mb reads (vs. ~1kb in other NGS methods)
Can sequence RNA

35. Nanopore “strand sequencing”
Watch this video:

36. Methods complementary to NGS
Goodwin et al., Coming of age: ten years of next generation sequencing technologies.
Nature Reviews Genetics, 17,

37. Microarrays detect presence/absence of defined motifs (not sequencing
Affimetrix microarrays

38. Microarrays and NGS are used for similar purposes, but there are differences in their applications:

39. Take home message
1st Generation Sequencing: one DNA molecule at a time
Maxam–Gilbert sequencing (“chemical sequencing”)
Sanger sequencing (“chain termination sequencing”)
Next Generation Sequencing: many DNAs in parallel
Rosh 454 (“pyrosequencing”) – already discontinued
Ion Torrent/Ion Proton (pH change) – cheap, but slow
Illumina Solexa (“sequencing by synthesis”, “bridge amplification”) – actively used in our school nowadays
Oxford Nanopore (“Nanopore sequencing”) – long reads, state of the art, also used in our school