GENOME SEQUENCING

BY :-
ANITHA.Y
14KUST4002
WHAT IS GENOMICS ?
GENOMICS AS A SUB DISCPLINARY
BRANCH

• MAPPING • SEQUENCING

• FUNCTIONAL
ANALYSIS OF GENOME
 DIFFERENCE B/W GENETICS
AND GENOMICS

looks at a single
gene where as
examines
all the genes of entire
system in a broader
manner.
• It deals with the Study of
functional and structural
aspects of genome aiding
in
.
STRUCTURAL-
Determination of complete
sequence and gene map.

FUNCTIONAL
FUNCTIONAL-
Functioning and
regulation of gene
STRUCTURAL COMPARATIVE
expression ; metabolic
pathway etc

COMPARATIVE- TYPES OF
GENOMICS
Compares genes from
different genomes to
relate functional and
“evolutionary
relationships”.
 GENOME
 IS DEFINED AS
.
OR

IN A E/K , DNA OR RNA OR

VIRUSES.
 IT IS DENOTED AS ‘X’.NUMBER OF PLOIDY OF AN ORGANISM.
 EXAMPLE :- DROSOPHILA MELANOGASTER (2n=2X=8) GENOME
:- X=4
 TRITICUM AESTIVUM (6n=6X=42) GENOME :- X=7
 THE TERM WAS
TO DENOTE COMPLETE SET OF CHROMOSOMAL AND
EXTRACHROMOSOMAL GENES IN ALL ORGANISM INCLUDING
VIRUS.
TYPES OF GENOME
PROKARYOTIC GENOME

EUKARYOTIC GENOME
• NUCLEAR GENOME
• MITOCHONDRIAL GENOME
• CHLOROPLAST GENOME
 Is a technique that allows researchers to read
the genetic information found in the DNA of
anything from bacteria to plant to animals.
 It basically involves “determination of order
of bases ”.
AGCTTAA CCCTGGTACCAAA GGGTCACTT
 Position of every gene along the
chromosome, regulatory gene that flank each
gene, the coding sequence that determines
the protein produce by each gene.
WHY SEQUENCE GENOME ? ? ?

IMPROVE
HUMAN HEALTH

HARNESS
CLEAN UP
NATURAL
ENVIRONMENT
ENERGY

ENSURE FOOD CONSERVE

SAFETY ENVIRONMENT
 WHY SEQUENCE GENOME ? ? ?
 DISTINGUISH B/W GENOMES OF FLORA AND FAUNA ,
AND THEN GROUP THEM TO WHICH FIELD THEY
BELONG.
 SERVES AS A MEANS OF CROP IMPROVEMENT AND
DEVELOP CONVENTIONAL BREEDING PRACTICES TO
DEVELOP AGRICULTURAL PRACTICES.
 IT CAN ALSO BE USED TO PRODUCE SUFFICIENT
AMOUNT OF SAFE AND NUTRITIOUS FOOD DURING
INCREASE POPULATION DEMAND.
 HELP RESEARCHES OR PRODUCERS (FARMERS) IN
DETERMINING THE RELATIONSHIP B/W STRESS AND
THE YIELD POTENTIAL THUS HELPING IN IMPROVING
VARIETIES.
MILESTONES
 1977- FIRST SEQUENCED Φx 174 BACTERIOPHAGE GENOME
(F.SANGER) (5,375bp)
 1995- HAEMOPHILUS INFLUENZAE (1,830,137bp) ; MYCOPLASMA
GENITALIUM (5,80,000bp) CRAIG VENTER AND HAMILTON SMITH)
 1996- SACCAROMYCES CEREVISIAE (12,068,000)
 1997- ESCHERICHIA COLI (4,639,221bp)
 2000- DROSOPHILA MELANOGASTER (180,000,000bp)
 2001- HUMAN WORKING DRAFT (320,000,000bp)
 2002-PLASMODIUM FALCIPARUM (23,000,000bp) ;ANOPHELES
GAMBIEA (278,000,000bp)
 2003- HUMAN FINISHED SEQUENCE (3,200,000,000bp)
 2005- ORYZA SATIVA (489,000,000bp)
 2006- POPULUS TRICHOCARPA (485,000,000bp)
SEQUENCING OF OTHER ORGANISM

 The sequence of many organisms have been carried

out at a rapid pace
 There are many medical ; genetics ; and commercial
reasons for sequencing genomes of other organisms
:-
 Escherichia coli
 Saccharomyces cerevisiae
 Caenorhabditis elegans
 Drosophila melanogaster
 Arabidopsis thaliana(Thale / Mouse ear cress)
 Oryza sativa L. (Rice)
 Mus musculus(laboratory mouse)
DETERMINING THE SEQUENCE OF DNA
 METHODS :-
 MAXAM & GILBERT CHEMICAL DEGRADATION METHOD
 FREDRICK SANGERS CHAIN TERMINATION METHOD
 GENOME SEQUENCING METHODS :-
 - SHOT GUN SEQUENCING
 - CLONE CONTIG APPROACH
 - AUTOMATED SEQUENCER
 - FLUORESCENCE SEQUENCER
 2nd GENERATION SEQUENCING METHOD :-
 - PYRO SEQUENCING
 - NANOPORE SEQUENCING
 - ILLUMINA SEQUENCING
 - SOLID SEQUENCING
 3rd GENERATION SEQUENCING METHOD
 AUTOMATED SEQUENCER
• First automated sequence was
invented by Llyod. M.Smith.
• It uses the Sangers sequence
method and formed the basis of
first generation of Dna sequences.
• The 1st automated sequencer was
AB370A which was able to
sequence 96 samples
simultaneously with 500-600kb in
size.
• Later in 1955 AB310 took over and
completed the ‘’ human genome
project “ in 2001
• The manufactures of this sequencer
are Roche , Illumina ,Life
Technologies ,Beckman
coulter,Pacific biosciences and
Oxford nanopore.
 AUTOMATED SEQUENCER
• It is basically radio active
sequencing approach.
• The electrophoretic bands gets
activated by scanning laser.
• The colour is read by and then
the computer assembles the
images as electropherograms
• They analyse the resulting
electropherograms giving the
output as four colour
chromatogram.
• There are many software tools
which are optimized for
sequencing the data like
preassemble ,seqtrace etc.
 FLUORESCENCE
• SEQUENCER
Since it was radio active in
nature there were disposable
problems and health risks
• Hence fluorescence sequencer
used fluorescent dyes .
• Sequencing products are
electrophoresed and they use
laser to detect fragments.
• Incorporation of randomly
labelled ddNTPs produces a
series of fragment in which
chain growth has been
terminated at each successive
position.
• Each nucleotide will be longer
than the previous one.
• Separation of fragments for
determining the order by size in
the form of ladder and as a
series of coloured band.
• Polyphred is a software tool.
As of september 2007, complete
sequence was known :

1879 viruses

577 bacterial species

Approx 23 E/K species (mostly half are viruses)

REFERENCES:-
 Brown B,Aoran M (2001) The rise of modern
genomics,3rd edn Wiley,New York,p234-295
 C.A Graham and A.J.M Hill, vol 167: Dna sequencing
protocols,p645-660
 Bishop, J.E & Waldholz M.Genome (Simon and
Schuster New york,1990),p109-117
 Aoran M The future of genomics ,proceedings of the
genomics researchers,boston (e-book)
 Springer
 Research gate