Genome Sequencing

• Goal
 figuring the order of nucleotides across a genome

• Problem
 Current DNA sequencing methods can handle only short stretches of DNA at once (<1-2Kbp)

• Solution
 Sequence and then use computers to assemble the small pieces

115
 A quick history of sequencing
• 1869 – Discovery of DNA
• 1909 – Chemical characterisation
• 1953 – Structure of DNA solved
• 1977 – Sanger sequencing invented
• – First genome sequenced – ФX174 (5 kb)
• 1986 – First automated sequencing machine
• 1990 – Human Genome Project started
• 1992 – First “sequencing factory” at TIGR
A quick history of sequencing
 Genome Sequencing
TG..GT TC..CC
AC..GC
CG..CA
TT..TC
TG..AC
AC..GC GA..GC
CT..TG
GT..GC AC..GC AC..GC
AA..GC AT..AT
TT..CC

Genome Short fragments of DNA Short DNA sequences

ACGTGGTAA CGTATACAC TAGGCCATA ACGTGACCGGTACTGGTAACGTACA

CCTACGTGACCGGTACTGGTAACGT
GTAATGGCG CACCCTTAG ACGCCTACGTGACCGGTACTGGTAA
CGTATACACGTGACCGGTACTGGTA
TGGCGTATA CATA… ACGTACACCTACGTGACCGGTACTG
GTAACGTACGCCTACGTGACCGGTA
CTGGTAACGTATACCTCT...
ACGTGGTAATGGCGTATACACCCTTAGGCCATA

Sequenced genome
118 118
 Manual Sanger Sequencing

• 800-1000 nucleotide

• Uses DNA polymerase

Principles

• Mix DNA with dNTPs and dideoxyNTPs

• Random termination at specific bases

• Separate by polyacrylamide gel electrophoresis

o Fragments migrate distance that is proportional to their size

119
 Sanger Sequencing

 DNA synthesis follow 5’ – 3’ direction

3’OH of sugar with 5’ of next sugar of following Nucleotide

 With ddNTPs – No 3’OH → stop DNA synthesis

Automated Sanger Sequencing
dideoxyNTPs
a person’s DNA
(all 3 billion bases)
can be sequenced
in a few days
Electrophoresis and gel reading:

 The reaction mixture from four batches are

loaded into four different well on gel

 The autoradiogram of the gel is read to

determine the order of bases of complementary
strand to that of template strand.

 The band of shortest fragments are at the

bottom of autoradiogram so that the sequences
of complementary strand is read from bottom to
top.
Sanger Sequencing

127
 Sanger Sequencing
• Advantages
 Long reads (~900bps)
 Suitable for small DNA fragments

• Disadvantages
 Low throughput
 Expensive
 Not suitable for long DNA fragments

130
 Sanger Sequencing
2007: Global Ocean Sampling
1994: H. Influenzae Expedition
1.8 Mbp ~3,000 organisms, 7Gbp (Venter et al.)
(Fleischmann et al.)

1980 1990 2000

1982: lambda virus 2001: H. Sapiens, D.

DNA stretches up to Melanogaster
30-40Kbp 3 Gbp
(Sanger et al.) (Venter et al.)

131
 Assembly: How Much DNA?
Input Output

Low coverage:

A few pieces to
assemble  many contigs, many
gaps 

High coverage:

many pieces to
assemble
 
a few contigs, a few
gaps

132
Lander and Waterman, 1988
 Next Genome Sequencing
1) Next Generation Sequencing (NGS) - An Introduction.mp4

• NGS is a general term referring to all post-Sanger sequencing technologies that enable massive
sequencing at low cost.
 Next-Generation Sequencing

 Pyrosequencing (454/Roche): detects pyrophosphate release using fluorescence,

after nucleotides are incorporated by polymerase to a new strand of DNA.

 Solexa/Illumina: works by simultaneously identifying DNA bases, as each base

emits a unique fluorescent signal, and adding them to a nucleic acid chain.

• Ion Torrent: sequencing measures the direct release of H+ (protons) from the
incorporation of individual bases by DNA polymerase and therefore differs from the
previous two methods as it does not measure light.
(454 sequencing) pyrosequencing
 pyrosequencing
+
pyrophosphate APS
(released by dNTP incorporation) adenosine
5`-phosphosulfate

ATP sulfurylase

+
sulfate
ATP
 pyrosequencing
O
+ 2
+
ATP oxygen luciferin

firefly luciferase

Light is positive correlated

with ATP used

AMP + pyrophosphate + light + oxyluciferin

 pyrosequencing
Pyrogram

Ronaghi M. Genome Res 11:3-11, 2001 Peaks in pyrogram reflex the Nucleotide sequence
 pyrosequencing
more biochemistry
problem solution
apyrase
free dNTP breaks down ATP to
AMP + 2 Pi
(or wash out solution)

use an analog suitable for

polymerase but not luciferase
luciferase can use dATP

dATPαS
 Application
 Human genome sequencing

 New virus and bacteria genome sequencing

 New mutation, especially in bacteria resistant to drug

 Analyze Single Nucleotide Polymophism

 Advantages
 400-600 mil bp in 10h, much faster than Sanger sequencing (tens of day)

 High accurency: 99.9% with 200 base-fragment and 99% with 400 base-
fragment.
 Disadvantages
 Difficult for repeat sequence: >6 Nucleotides

 Complex, require many steps

 High reagent cost

 Illumina sequencing

Sequencing by synthesis
Sample preparation
Fluorescence beam
Nanopore – 3rd generation sequencing

For long read DNA, RNA, protein

Without the need of DNA amplification

Nanopore protein

Electrically resistant membrane

 Electric field apply,
DNA moving

Voltage apply

Measure magnetic current across nanopore

Thank you
Questions?