Genome sequencing and comparative genomics are important tools in plant breeding. Genome sequencing determines the order of DNA nucleotides in individual genes, chromosomes, or entire genomes. Comparative genomics analyzes and compares genetic material between species to study evolution, gene function, and disease. Next generation sequencing techniques like Illumina sequencing have made genome sequencing faster, cheaper, and able to sequence thousands of sequences at once. Comparative genomics is used to understand differences between species by comparing gene location, structure, sequence similarity and other characteristics. This aids in understanding evolution and identifying genes responsible for unique traits.

1. genome SEQUENCING &comparative genomics
ANJANI KUMAR
Registration No:- A/BAU/5129/2017
Sub:- Genomics in Plant Breeding (GP603)
Department of Genetics and Plant Breeding
FACULTY OF AGRICULTURE
BIRSA AGRICULTURAL UNIVERSITY
KANKE, RANCHI – 834 006 (JHARKHAND)

2. WHAT IS A GENOME?
• In Drosophila melanogaster (2n = 2x = 8); genome x = 4.
• In hexaploid Triticum aestivum (2n = 6x = 42); genome x = 7.
• It is denoted by x. Genome depends on the number of ploidy of
organism.
IS DEFINED AS “WHOLE HEREDITARY
INFORMATION OF AN ORGANISM THAT IS
ENCODED IN THE DNA”
.
The term genome was introduced by H. Winkler in
1920 to denote the complete set of chromosomal
and extra chromosomal genes present in an
organism, including a virus.
In cytogenetic genome means a single set of
chromosomes.

3. TYPES OF GENOME
PROKARYOTIC GENOME
EUKARYOTIC GENOME
NUCLEAR GENOME
MITOCHONDRIAL GENOME
CHLOROPLAST GENOME

4. CEREAL GENOME SIZES
 Sorghum 1000 Mb
 Maize 3000 Mb
 Barley 5000 Mb
 Wheat 16,000 Mb
 Rice 420 Mb

5. WHAT IS GENOMICS ?

6. GENOMICS AS A SUB DISCPLINARY BRANCH
MAPPING SEQUENCING
FUNCTIONAL ANALYSIS OF
GENOME

7. DIFFERENCE B/W GENETICS
AND GENOMICS
 Genetics looks at a single gene
where as Genomics 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
interaction of genes with the
loci and alleles.

8. STRUCTURAL
Determination of complete
sequence and gene map.
FUNCTIONAL
Functioning and
regulation of gene
expression ; metabolic
pathway etc
COMPARATIVE
Compares genes from
different genomes to
relate functional and
“evolutionary
relationships”.
TYPES OF
GENOMICS
STRUCTURAL
FUNCTIONAL
COMPARATIVE

9. • Genome sequencing is figuring out the order of DNA nucleotides.
• Used to determine the sequence of individual genes, larger genetic
regions, full chromosomes or entire genomes.
• The resulting sequences may be used by researchers in molecular biology.
GENOME SEQUENCING

10. • Deciphering “code of life”
• Detecting mutations
• Typing microorganisms
• Identifying human haplotypes
• Designating polymorphisms
PURPOSE

11. The sequencing of DNA molecules
began in the 1970s with development
of the Maxam-Gilbert method, and
later the Sanger method.
Originally developed by Frederick
Sanger in 1975, most DNA sequencing
that occurs in medical and research
laboratories today is performed using
sequencers employing variations of
the Sanger method.
HISTORY OF GENOME SEQUENCING

13. DNA STRUCTURE
In a strand of DNA, there are some simple
units known as nucleotides. These
nucleotides have a 'backbone' that
consists of sugars and a phosphate group.
The DNA bases can be one of four kinds
and they are attached to these sugars.
These bases hold the important and
unique genetic information for body.
These bases are:
• Adenine (A)
• Thymine (T)
• Cytosine (C)
• Guanine (G)

14. Methods of sequencing
1. Sanger dideoxy (primer extension/chain-termination)
method: most popular protocol for sequencing, very
adaptable, scalable to large sequencing projects
2. Maxam-Gilbert chemical cleavage method: DNA is labelled
and then chemically cleaved in a sequence-dependent
manner. This method is not easily scaled and is rather
tedious
3. Shotgun sequencing
4. 454 pyrosequencing
5. Ion Torrent semiconductor sequencing
6. Illumina (Solexa) sequencing

15. • Most common
approach used for
DNA sequencing
• Invented by Frederick
Sanger - 1977
• Nobel prize - 1980
• Also termed as Chain
Termination or
Dideoxy method
1. SANGER METHOD

16. DEOXY VERSUS DIDEOXY

17. Chain Termination (Sanger) Sequencing
The 3′-OH group necessary for formation of the phosphodiester bond
is missing in ddNTPs.
Chain terminates at ddG

18. • Chain termination method of DNA sequencing.
• It involves following components:
Primer
DNA template
DNA polymerase
dNTPs(A,T,G,C)
ddNTPs
• 4 Steps:
1. Denaturation
2. Primer attachment and extension of bases
3. Termination
4. Poly acrylamide gel electrophoresis
Chain Termination (Sanger) Sequencing

19. Chain Termination (Sanger) Sequencing
• With addition of enzyme (DNA polymerase), the primer
is extended until a ddNTP is encountered.
• The chain will end with the incorporation of the ddNTP.
• With the proper dNTP:ddNTP ratio, the chain will
terminate throughout the length of the template.
• All terminated chains will end in the ddNTP added to
that reaction.

20. Chain Termination (Sanger) Sequencing

21. 2. MAXAM & GILBERT METHOD
• A. M. Maxam and
W.Gilbert-1977
• Chemical Sequencing
Treatment of DNA with
certain Chemicals - DNA
cuts into Fragment
Monitoring of
sequences

22. Maxam-Gilbert sequencing is performed by chain
breakage at specific nucleotides by chemical reagent

23. • Shotgun sequencing, also known as shotgun cloning, is a method used
for sequencing long DNA strands or the whole genome.
•In shotgun sequencing, DNA is broken up randomly into numerous small
segments and overlapping regions are identified between all the individual
sequences that are generated.
• Multiple overlapping reads for the target DNA are obtained by
performing several rounds of this fragmentation and sequencing.
•Computer programs then use the overlapping ends of different reads to
assemble them into a continuous sequence.
•The shotgun approach was first used successfully with the bacterium
Haemophilus influenzae.
•Craig Venter used this method to map the Human genome project in 2001.
Shotgun sequencing

24. Mechanism of shotgun

25. Disadvantages of Shotgun sequencing
• Overlapping sequencing is missing
• Repetitive DNA sequence is not sequenced
Advantage of Shotgun sequencing
• Whole genome sequence
• Less time
• Accurate

27. Next Generation Sequencing (NGS)
• High throughput DNA Sequencing Technique
• Employs Micro and Nanotechnologies
• Reduce sample size
• Low Reagent cost
• Less Time
• Massive Parallel Sequencing
• Sequence thousands of sequences at once
• Produce enormous amount of data

28. Next Generation Sequencing (NGS)
454 sequencing
Ion torrent sequencing
Illumina sequencing

31. Pyrosequencing/454 sequencing

32. Pyrosequencing/454 sequencing
• Pyrosequencing is based on the generation of
light signal through release of pyrophosphate
(PPi) on nucleotide addition.
– DNAn + dNTP  DNAn+1 + PPI
• PPi is used to generate ATP from adenosine
phosphosulfate (APS).
– APS + PPI  ATP
• ATP and luciferase generate light by conversion of
luciferin to oxyluciferin.

33. Methodology
1. DNA fragmentation
2. Adapter binding to ends of DNA fragments.
3. Denaturation of fragments
4. Fix to a solid surface, Sepharose beads or
streptavidin-coated magnetic beads
5. Beads has sequences complementary to
adapter sequences.

34. Load the beads into sequencing wells (small DNA grooves).
Chemical process
I. Add DNA polymerase and single dNTP (polymerization)
II. Add sulfurylase and APS ( ATP synthesis)
III. Add luciferin and luciferase (light production)
IV. Detection ( light sensor)
V. Washing or add enzyme apyrase enzyme.
Contd…..

36. Chemistry of Pyrosequencing

37. Application of Pyrosequencing
• Whole genome sequencing
• Targeted resequencing
• Sequencing-based Transcriptome
Analysis
• Metagenomics
• mutation detection
• viral typing, bacterial typing, and SNPs.

38. • Accurate
• Parallel processing
• Easily automated
• No need for gel electrophoresis
• Nonlinear light response after more than 5-6 identical
nucleotides
Advantage of Pyrosequencing
Disadvantages of Pyrosequencing

42. Application of Ion torrent sequencing
 Whole genome sequencing
 It takes only 2-3hrs
 Very fast
 Not complicated
 No fluorescence requered

44. Diagram overview

50. Denature the Double Stranded Molecules
• The original strand is then washed
away, leaving only the strands that
had been synthesized to the oligos
attached to the flow cell

53. Image First Base
• Remove unincorporated bases
• Detect Signal
• Deblock and remove the fluorescent
signal-new cycle

54. Determine Second Base
• Add sequencing reagents
• Primers
• Polymerase
• Fluorescently labeled nucleotide
• Buffer
• Second base incorporated

55. • Remove unincorporated bases
• Detect Signal
• Deblock and remove the
fluorescent signal - new cycle
Image Second Chemistry Cycle

56. Align Data
• After the sequencing is
finished they are aligned
• Each was once one larger
sequence that had been
fragmented
• Needs to be realigned to
find the original sequence of
the larger sequence

57. • DNA sequencing
• Gene Regulation Analysis
• Sequencing-based Transcriptome Analysis
• SNPs discovery
• Cytogenetic Analysis
• Small RNA discovery analysis
Application of Illumina sequencing

60. APPLICATIONS OF NGS
• Mutation discovery
• Transcriptome Analysis – RNA-
Seq
• Enabling Metagenomics
• Defining DNA-Protein interactions
– ChIP-Seq
• Discovering non-coding RNAs
• Molecular diagnostics for
Oncology & Inherited Disease
study
• Gene Regulation Analysis
• Whole Genome Sequencing
• Exploring Chromatin Packaging

62. WHAT IS COMPARATIVE GENOMICS?
 Analyzing & comparing genetic material from different
species to study evolution, gene function, and inherited
disease
 To understand the uniqueness between different species

63. COMMONLY USED TERMS
PARALOGS ARE GENES FOUND IN THE SAME
SPECIES THAT WERE CREATED THROUGH
GENE DUPLICATION EVENTS
ORTHOLOGS ARE GENES FOUND IN TWO
SPECIES THAT HAD A COMMON ANCESTOR

64. WHY WE MAKE COMPARISON
 Comparative genomics is a field of biological research in
which the genome sequences of different species —
human, mouse, and a wide variety of other organisms from
bacteria to chimpanzees — are compared.
 By comparing the sequences of genomes of different
organisms, researchers can understand what, at the
molecular level, distinguishes different life forms from each
other.
 Comparative genomics also provides a powerful tool for
studying evolutionary changes among organisms, helping
to identify genes that are conserved or common among
species, as well as genes that give each organism its unique
characteristics.

65. WHAT IS COMPARED?
 Gene location
 Gene structure
 Exon number
 Exon lengths
 Intron lengths
 Sequence similarity
 Gene characteristics
 Splice sites
 Codon usage

69. HOMOLOGY SEARCHES
BLAST

70. Gramene (http://www.gramene.org) is a
comparative genome mapping database for
grasses and a community resources for rice
Gramene

71. Rice
 Whole genome sequence
Other crop grasses
Maize
Sorghum
Millet
Sugarcane
Wheat
Oat
Barley
Gramene scope

72. ANTICIPATED BENEFITS OF GENOME RESEARCH
Forensics: to help identify individuals
because each individual has a
different genetic sequence
Medicine: can be used to help detect
the genes which are linked to various
genetic disorders such as muscular
dystrophy.
Agriculture: The mapping and
sequencing of a genome of
microorganisms has helped to make
them useful for crops and food
plants.

73. FUTURE PROSPECTS

74. Thank
you