Systems Microbiology
Monday Oct 23 - Ch 15 -Brock
Genomics
•
•
•
•
DNA sequencing technology
Genome sequencing technology
Current statistics
Basics of genome sequence analysis
Human genome sequence analysis reached a first stage of completion in the summer of 2000 summer by the New York Times:
Scanned magazine and newspaper articles about J. Craig Venter
and Francis Collins removed due to copyright restrictions.
The Race to Sequence
Cartoon image of J. Craig Venter and Francis Collins
racing to the finishremoved due to copyright restrictions.
• Celera - J. Craig Venter
• NHGRI - Francis Collins
• G5
–
–
–
–
–
Sanger Centre
Whitehead Institute
JGI Walnut Creek
Washington University
Baylor
Human Genome Project
• HGP drove innovation in biotechnology
• 2 major technological benefits
– stimulated development of high throughput methods -- the
assembly line (or dis-assembly line) meets biological research
– reliance on computational tools for data mining and
visualization of biological information
DNA Sequencing
• DNA sequencing = determining the nucleotide sequence of DNA.
• Developed by Frederick Sanger in the 1970s.
First whole DNA genome sequenced
was a virus, PhiX174 in 1977 - 5386 bp.
First demo of Sanger
dideoxynucleotide nucleotide
sequencing technique.
Photographs removed due to copyright restrictions.
1980: Walter Gilbert (Biol. Labs) & Frederick Sanger (MRC Labs)
Manual Dideoxy DNA sequencing-How it works:
1. DNA template is denatured to single strands.
2. DNA primer (with 3’ end near sequence of interest) is annealed to the
template DNA and extended with DNA polymerase.
3. Four reactions are set up, each containing:
1.
2.
3.
4.
DNA template
Primer annealed to template DNA
DNA polymerase
dNTPS (dATP, dTTP, dCTP, and dGTP)
4. Next, a different radio-labeled dideoxynucleotide (ddATP, ddTTP, ddCTP, or
ddGTP) is added to each of the four reaction tubes at 1/100th the
concentration of normal dNTPs.
5. ddNTPs possess a 3’-H instead of 3’-OH, compete in the reaction with normal
dNTPS, and produce no phosphodiester bond.
6. Whenever the radio-labeled ddNTPs are incorporated in the chain, DNA
synthesis terminates.
7. Each of the four reaction mixtures produces a population of DNA molecules
with DNA chains terminating at all possible positions.
Manual Dideoxy DNA sequencing-How it works (cont.):
8. Extension products in each of the four reaction mixutes also end with a
different radio-labeled ddNTP (depending on the base).
9. Next, each reaction mixture is electrophoresed in a separate lane (4 lanes) at
high voltage on a polyacrylamide gel.
10. Pattern of bands in each of the four lanes is visualized on X-ray film.
11. Location of “bands” in each of the four lanes indicate the size of the fragment
terminating with a respective radio-labeled ddNTP.
12. DNA sequence is deduced from the pattern of bands in the 4 lanes.
5’
C
O
O-
3’
O
P
O
C
OH
H
primer
template
Sanger sequencing works by primer extension using
DNA polymerase and the 4 deoxynucleotide triphosphates
PLUS one dideoxynucleotide per sequencing rxn.
Newly synthesized DNA
5’
O
O-
3’
O
P
O
C
X
OH
H
3’ dideoxynuclotide
Images of polyacrylamide gels removed due to copyright restrictions.
Vigilant et al. 1989
PNAS 86:9350-9354
(polyarcrylamide gel)
Radio-labeled ddNTPs (4 rxns)
Sequence (5’ to 3’)
G
G
A
T
A
T
A
A
C
C
C
C
T
G
T
Long products
Short products
Automated Dye-Terminator DNA Sequencing:
1. Dideoxy DNA sequencing was time consuming, radioactive, and throughput
was low, typically ~300 bp per run.
2. Automated DNA sequencing employs the same general procedure, but uses
ddNTPs labeled with fluorescent dyes.
3. Combine 4 dyes in one reaction tube and electrophores in one lane on a
polyacrylamide gel or capillary containing polyacrylamide.
4. UV laser detects dyes and reads the sequence.
5. Sequence data is displayed as colored peaks (chromatograms) that
correspond to the position of each nucleotide in the sequence.
6. Throughput is high, up to 1,200 bp per reaction and 96 reactions every 3
hours with capillary sequencers.
7. Most automated DNA sequencers can load robotically and operate around the
clock for weeks with minimal labor.
Applied Biosystems PRISM 377
(Gel, 34-96 lanes)
Applied Biosystems PRISM 3700
(Capillary, 96 capillaries)
Photographs of equipment removed due to copyright restrictions.
Applied Biosystems PRISM 3100
(Capillary, 16 capillaries)
“virtual autorad” - real-time DNA sequence output from ABI 377
1. Trace files (dye signals) are analyzed and bases called
to create chromatograms.
2. Chromatograms from opposite strands are reconciled
with software to create double-stranded sequence
data.
Table and graph showing the growth of GenBank removed due to copyright restrictions.
Fraser, Eisen, and Salzman
Shotgun genome sequencing
Nature 406, 799-803 (17 August 2000)
Images removed due to copyright restrictions.
Small insert cloning/seqing
Image removed due to copyright restrictions.
See Figure 15-1a in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.
11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.
Blue white selection of insert containing clones
based on lacZ (beta galactosidase) activity
Image removed due to copyright restrictions.
See Figure 15-1bc in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.
11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.
{
Fluorescent Sanger dideoxy seqing rxns,
& capillary electrophoresis !
Reads
Contigs
Genome
Figure by MIT OCW.
(typically, shotgun seqing done to 8X min.)
Shotgun sequencing: A Bottom Up, Random Strategy
STS
Mapped Scaffolds:
Genome
Scaffold:
Read pair (mates)
Contig:
Gap (mean and standard deviation known)
Consensus
Reads (of several haplotypes)
SNPs
BAC Fragments
Figure by MIT OCW.
The key to the strategy is shotgun cloning of random (not mapped) fragments of a defined length (2kb or 10kb)
so that scaffolds can be assembled from terminal sequences of each cloned insert.
DNA Sequence Assembly
Computer screenshot showing DNA sequencing removed due to copyright restrictions.
Large insert cloning/seqing
Image removed due to copyright restrictions.
See Figure 15-2 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.
11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.
BAC sequencing: A Top down Up Strategy Using Maps
Hierarchial shotgun sequencing
Genomic DNA
BAC library
Organized mapped
large clone contigs
BAC to be sequenced
Shotgun clones
...ACCGTAAATGGGCTGATCATGCTTAAA
TGATCATGCTTAAACCCTGTGCATCCTACTG..
...ACCGTAAATGGGCTGATCATGCTTAAACCCTGTGCATCCTACTG...
Shotgun
sequence
Assembly
Figure by MIT OCW.
BAC = Bacterial Artificial Chromosome (F-Factor based)
Image removed due to copyright restrictions.
The NIH consortium strategy was dependent on multiple clone overlaps
Brief technical outline for
DNA sequence production
High throughput DNA sequencing is entirely automated from colonies to computers
Approach made possible by automating massively parallel processes on an industrial scale
Photograph of an industrial scale DNA sequencing lab removed due to copyright restrictions.
Sequence Production Pipeline
_ Pick & archive library clones
_ Template production
_ dideoxy terminator reactions
_ Post-reaction processing
_ Capillary sequencing
_ Sequence data analysis - bioinformatics
Making the Genome Bite Size
Image removed due to copyright restrictions.
Making a DNA fragment library
LacZ gene produces
blue pigment
Sheared fragments
pUC18
The Lacz gene is
disrupted by the
insert
Recombinant
DNA
Gene for antibiotic
resistance
Hybidization + DNA ligase
A couple of thousand inserts
are created from each original
large sequence. Each color is a
different sheared fragment
Some of these
plasmids do not
take in a fragment
Figure by MIT OCW.
Plating a DNA Fragment Library
Plating photographs removed due to copyright restrictions.
Picking & Archiving Clones
• QPixII from Genetix, Ltd (UK)
• 4500 colonies per hour
• 300,000 colonies; 67 hrs; 384-well plates = 781
Various laboratory photographs removed due to copyright restrictions.
454
Corporation
The first commercial,
massively parallel,
DNA sequencing technology
Images removed due to copyright restrictions.
Deposit DNA Beads into PicoTiterPlate™
Images of DNA beads on PicoTiterPlates removed due to copyright restrictions.
Sequencing Instrument
Images removed due to copyright restrictions.
454 DNA sequencing
technology
– 300,000 DNA fragments sequenced in parallel
– Average read length 100 - 110 nt
– 30 million nt per run
– One 454 run = 4 hours
– $5,000 reagent cost
Company
Format
Read length
Throughput
Sequencing
Amersham
Capillary electrophoresis
550-1,000 bases
2.8 megabases/day
(384-capillary instrument)
Applied Biosystems
(Foster City, CA)
Capillary electrophoresis
500-950 bases
(depending on
column length)
2 megabases/day
Lynx Therapeutics
(Hayward, CA)
Massively parallel
bead arrays
20 bases
8 megabases/day
Pyrosequencing
(Uppsala, Sweden)
Real-time sequencing
by synthesis
40-50bases/well
(96- or 384-well plate)
110-691 kilobases/day
454 Life Sciences
(Branford, CT)
Massively parallel
microfluidic, solid-surface
sequencing
100 bases currently
60 megabases/day
Affymetrix
(Santa Clara, CA)
High-density microarrays
30 kilobases/array currently;
500 kilobases/array projected
3 megabases/day
Perlegen
(Mountain View, CA)
Whole-wafer Affymetrix
15 megabase/wafer
300 megabases/day
Biosciences
(Uppsala, Sweden)
(production-scale system)
Resequencing
microarray
Sequencing and resequencing technologies currently available
Figure by MIT OCW.
http://www.genomesonline.org/Gold_statistics.html
Courtesy of Genomes Online. Used with permission.
http://www.genomesonline.org/Gold_statistics.html
Courtesy of Genomes Online. Used with permission
http://www.genomesonline.org/Gold_statistics.html
Courtesy of Genomes
Online. Used with permission.
_____________
2
xis
(5
rgi
s
OP11
acteriu
m (1)
Nitrospira
bia
Vernicomicro
25
(62
)
ca
Aquili
les
KORARCHAEOTA
ha
no
co
s3
lfobacterium
Thermodesu 1
et
cu
Thermotogales 1
M
oc
Dictyo
OP9
g
Coprochemobacte lomus
r
(1)
Ce
es
chaeot
renar
tonic C
plank
planktonic mesophiles
oc
on
-su
m
aeu
ch
nar
Therm
erm
Gr
ee
nn
0
P8
pJ 23
22
L1 7
pS SL 1 pSL L 4
p
pS 78
pSL
2
1
L
pS
Th
A
3
ac
b
no
cti
ia
ter
2)
(1
78
pJP
pJP 27
10
OP 1
S
W
OP5
)
s (1
ete
myc
CRENARCHAEOTA 4 (3)
s1
opyru rmus
n
a
h
t
Me thanothe
acterium 1 EURYARCHAEOTA 12(4)
Me
Methanob
Archaeoglobus 1
OP3
lfu
r
myd
to
OS-K
2)
ia 5 (
Chla
c
Plan
ia
ter
0
L5
pS
pJP41
tip )
es
tes
Term
OP
ite g
roup
8
Acidob
1
ne
Pyrobaculum 1
Hyperthermus (1)
Aero
Su pyr
u
lfo
lob m 1
us
2(
2)
ia
Fle
1)
16
ter
ARCHAEA
16 (7)
Thermoblum
ive
ur (
sit
Fib
po
Sy
ac
ba
c
am
gr
ob
sulf roup A
en
g
r
Gre
ine cte
r
a
2)
Ma rob
(3
C
an
TM6
WS
TM76
Fus
oba
Pr
cter
ote
ia
o
G+
Cy
BCF group (5)
1 (1)
Thermus/Deinococcus
Spirochetes 2 (2)
w
Lo
BACTERIA
56 (125)
cc
us
oplasm
a 2 (1)
Meth
anos
arcin
a 2 (1
M
1
(1
)
eth
Ha
ano
lo
ph
ile
s1
)
spir
illu
m
(1
)
EUCARYA
Figure by MIT OCW.
Courtesy of Genomes Online. Used with permission.
http://www.genomesonline.org/Gold_statistics.html
Courtesy of Genomes Online. Used with permission.
http://www.genomesonline.org/Gold_statistics.html
Courtesy of Genomes Online. Used with permission.
Tables removed due to copyright restrictions.
See Tables 15-1 and 15-3 in Madigan, Michael, and John Martinko.
Brock Biology of Microorganisms. 11th ed. Upper Saddle River, NJ:
Pearson Prentice Hall, 2006. ISBN: 0131443291.
Genome Streamlining in a Cosmopolitan Oceanic Bacterium
Giovanonni et al, Science 309:1242 (2005)
10.0
Streptomyces coelicolor
Rhodopirellula baltica
5.0
Genome size (Mbp)
Silicibacter pomeroyi
Coxiella burnetii
Bartonella henselae
Thermoplasma acidophilum
Bartonella quintana
Ehrlichia ruminantium
Prochlorococcus marinus MIT9313
Prochlorococcus marinus SS120
Prochlorococcus marinus MED4
Pelagibacter ubique
Rickettsia conorii
1.0
0.5
Synechococcus sp.WH8102
Mesoplasma florum
Mycoplasma genitalium
Wigglesworthia glossinidia
Nanoarchaeum equitans
Host-associated
Free-living
Obligate symbionts/parasites
Pelagibacter ubique
0.1
100
500
1000
5000
10000
Number of protein encoding genes
Figure by MIT OCW.
Science 314:267
(Oct 13, 2006)
70
GC content (%)
60
50
Carsonella
40
30
20
Carsonella
50 mm
10
0
0
1
2
3
4
5
6
7
8
9
Chromosomal size (Mb)
Figure by MIT OCW.