University of Leicester –Genomes–Microbial Genomics -October 2010 Page 1 Genomics-sequencing of microbial genomes This lecture illustrates the strategies used in microbial genome sequencing projects, compares genome content and organisation amongst microbes, and shows how to derive information on gene function across genome. Objectives for students: Expected to describe strategies involved in microbial genome sequencing and functional genomics Provide examples of information that can be derived from genomics Microbial Genome Sequencing Genome Sequencing Projects o strategy & methods o annotation Comparative genomics o organisation o gene content Functional genomics o transcriptome o proteome o genome-wide mutation Concentrate on strategy & ideas Genome Sequencing Projects Genome sequencing progress (2009) Complete: o Archaeal: 70 (2007 = 49) (2008= 55) o Bacterial: 945 (2007 = 554) (2008= 728) o (Eukaryotc : 121) (2007 = 76) (2008= 97) Ongoing: o Archaeal: 111 o Bacterial: 3498 o (Eukaryotic: 1223) o Metagenome projects: 200
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University of Leicester –Genomes–Microbial Genomics -October 2010 Page 1
Genomics-sequencing of microbial genomes
This lecture illustrates the strategies used in microbial genome sequencing projects, compares
genome content and organisation amongst microbes, and shows how to derive information on gene
function across genome.
Objectives for students:
Expected to describe strategies involved in microbial genome sequencing and functional
genomics
Provide examples of information that can be derived from genomics
In the pre-genome era there were a number of considerations regarding the benefits of sequencing.
The piecemeal collection of sequenced genes was slow and costly. Issues also arose over ownership,
strain choice, approach and data release. The genome project, however, provided a rational
approach to sequencing which was efficient and rapid, and was able to address novel questions. The
post genomic era has allowed the application of comparative and functional genomics.
Genome sequencing strategy:
Strategy choice
o large collaborative cosmid/BAC-based projects
now better suited for larger genomes
slow
o small insert shotgun approach
centralised
rapid and efficient
choice for bacteria
Strain choice
o fresh isolate vs lab strain
o clinical vs environmental
o subsequent genetic analysis
E.g. Yeast genome sequence strategy
Yeast chromosomes (16) individually sequenced
several approaches used
o Make genome library in cosmids
order cosmid library
need to know which cosmid overlaps with which
link cosmid to genome map
University of Leicester –Genomes–Microbial Genomics -October 2010 Page 4
produced tiled set of cosmids
only sequence minimum number
o Use chromosome specific probe to identify chromosome-specific cosmids
o sequence cosmid inserts by subcloning
o Solve problems by direct PCR sequencing, walking and other libraries (lambda)
o Telomeres
University of Leicester –Genomes–Microbial Genomics -October 2010 Page 5
Whole genome/chromosome shot-gun strategy (WGS)
Rapid
Generation of small insert genomic library
Library is not initially ordered
DNA sequence ends of inserts
Depends on powerful computing to assemble sequence reads
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Main steps in generating a complete genome sequence
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Automated sequencers:
Manually chain termination sequencing requires four reaction tubes each containing a different type of terminator base as well as a radioactive nucleotide for labelling the newly synthesised DNA fragments. Each of the four reactions is electrophoresed in a separate lane of a gel. Demand for the ability to read more sequence in a shorter amount of time, led to the automation of the DNA sequencing process.
The attachment the of different fluorescent dyes to each of the four terminator bases ensured four separate sequencing reactions were no longer required; the entire sequencing reaction could be accomplished in a single tube. The development of these automated sequencing machines using multiple capillaries, thin, hollow glass tubes filled with a gel polymer, removed the need for a technician to add each sequencing reaction into an individual lane of the gel prior to the run
ABI 3700
The ABI 3700s (made by Applied Biosystems) are the most widely used automated sequencers. They
have 96 capillaries, with a robot loading from 384-well plates.
MegaBACE
The MegaBACE is made by Amersham. It also has 96 capillaries and robotic loading from 384–well
plate. Each run takes two to four hours, and can read up to 800 bases.
These advances have lead to the industrialization of sequencing. Most genome sequencing projects
divide tasks (such as genome libraries, production sequencing and finishing) among different teams.
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Sequencing machines run are run 24 hours a day, 7 days a weeks and many tasks can be perfomed
by robots.
454 sequencing- the future?
454 sequencing was developed Roche, and relies on a technique known as pyrosequencing
(sequencing by synthesis). It differs from Sanger sequencing, relying on the detection of
pyrophosphate release on nucleotide incorporation, rather than chain termination with
dideoxynucleotides.
Nucleotides are flowed sequentially in a fixed order across the PicoTiterPlate device during a sequencing run.
During the nucleotide flow, hundreds of thousands of beads each carrying millions of copies of a unique single-stranded DNA molecule are sequenced in parallel.
If a nucleotide complementary to the template strand is flowed into a well, the polymerase extends the existing DNA strand by adding nucelotide(s).
Addition of one (or more) nucleotide(s) results in a reaction that generates a light signal that is recorded by the CCD camera in the instrument.
The signal strength is proportional to the number of nucleotides incorporated in a single nucelotide flow.
The GS FLX System software tracks the location of DNA carrying beads on a XY axis. Each bead
corresponds to a XY-coordinate on a series of images. The signal intensity per nucleotide flow is
recorded for each bead over time and is plotted to generate a flowgram. Each 10 hour sequencing
run on the GS FLX Titanium series will typically produce over one million flowgrams, one flowgram
per read.
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The development and impact of 454 sequencing. http://www.ncbi.nlm.nih.gov/pubmed/18846085
Rothberg et al.Biotechnology. Volume 26, 1117-1124 9/10/2008