Download Genome Sequencing Using a Mapping Approach

Lecture 15 – Gene
Cloning
Based on Chapter 08 Genomics: The Mapping
and Sequencing of Genomes
Copyright © 2010 Pearson Education Inc.
1. Introduction
10. Artificial Chromosomes
13. Genomic Libraries
14. Size Selection of DNA Fragments using
Agarose Gel Electrophoresis
Slide 15 - DNA Sequencing and Analysis of
DNA Sequences
DNA Sequencing
Fig. 08.19
Dideoxy DNA
sequencing of a
theoretical DNA
fragment
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
A G C T
Automated Sequencers
Pyrosequencing
Sequencing Genomes
Structural Genome Biologists use two general
approaches to sequencing whole genomes:
• The mapping approach – first used, slower,
but informs you of problems.
• The Whole Genome“Shotgun” approach –
rapid, but requires greater computational
expertise. Works well on smaller genomes
with low complexity.
Genome Sequencing Using a Mapping
Approach
• Genetic mapping
a.Cytogenetic maps
b.FISH maps
Genome Sequencing Using a Mapping
Approach
• Genetic mapping
a.Cytogenetic maps
b.FISH maps
c.Radiation Hybrid maps
Others to be discussed later
a.RFLP maps
b.SNP maps
c.STR maps
d.VNTR maps
Genome Sequencing Using a Mapping
Approach
•
•
•
•
Identify and genetically map marker genes.
Break genome up into BAC or YAC clones
Construct a Clone Contig Map
Sequence each BAC or YAC and locate the
markers in the sequences.
• Order the BACs or YACs to make a genomic
sequence.
a.Cytogenetic maps
Genome Sequencing Using a Mapping
Approach
Genome Sequencing Using a
Mapping Approach
Ultimately through the use of these
techniques a high density physical map
of sequence polymorphisms can be
generated and used as the basis for
squencing the genome.
Genome Sequencing Using a Whole-Genome
Shotgun Approach
1. The shotgun approach obtains a genomic sequence by
breaking the genome into overlapping fragments for
cloning and sequencing.
2. A computer is then used to assemble the genomic
sequence.
3. Advances that have made this approach practical for
large genomes include:
a. Better computer algorithms for assembling
sequences.
b. Automation in the actual sequencing.
c. Faster automated sequencers that sequence millions
of sequences at one time.
Genome Sequencing Using a Whole-Genome
Shotgun Approach
Figure 10.2. The direct shotgun
approach to obtaining the
genomic DNA sequence of an
organism.
Genome Assembly and Gene Prediction
FEATURES Location/Qualifiers
source 1..9965640
/organism="Candidatus Solibacter usitatus Ellin6076" /mol_type="genomic DNA"
/strain="Ellin6076"
/db_xref="taxon:234267"
gene
89..1402
/locus_tag="Acid_0001"
gene
1637..2755
/locus_tag="Acid_0002"
gene
2781..5228
/locus_tag="Acid_0003"
gene
complement(5232..6827)
/locus_tag="Acid_0004"
gene
complement(6844..8496)
/locus_tag="Acid_0005"
gene
8627..9400 /
locus_tag="Acid_0006"
gene
9397..10986
/locus_tag="Acid_0007"
gene
10983..11723
/locus_tag="Acid_0008"
gene
12211..12804
/locus_tag="Acid_0009"
gene
12823..12996
/locus_tag="Acid_0010"
gene
complement(13323..13550)
/locus_tag="Acid_0011“
gene
complement(13560..17015)
/locus_tag="Acid_0012"
gene
complement(17148..19577)
/locus_tag="Acid_0013"