INTRODUCTION WHY SEQUENCING GENOME? SHOTGUN SEQUENCING HISTORY PRINCIPLE STEPS IN SHOTGUN SEQUENCING STRATEGIES FOR SHOTGUN SEQUENCING 1. Hierarchical shotgun sequencing 2. Whole genome shotgun sequencing MILESTONE IN GENOME SEQUENCING CONCLUSION REFERENCES

1. Genome sequencing
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. SYNOPSIS
 INTRODUCTION
 WHY SEQUENCING GENOME?
 SHOTGUN SEQUENCING
 HISTORY
 PRINCIPLE
 STEPS IN SHOTGUN SEQUENCING
 STRATEGIES FOR SHOTGUN SEQUENCING
1. Hierarchical shotgun sequencing
2. Whole genome shotgun sequencing
 MILESTONE IN GENOME SEQUENCING
 CONCLUSION
 REFERENCES

3. INTRODUCTION
 Genomics, the field dedicated to the study of genome data, is at the
heart of a revolution in genetic and medical research.
 With an ever increasing pace, whole genomes, the complete set of
genetic instructions for an organism, are being read and recorded for
creatures great and small.
 The sequencing of the human genome, over 3 billion nucleotides in
length, is already considered to be one of humankind’s greatest
achievements.
 This is due to advances in technology that enabled the application of
new techniques that greatly accelerated the process of reading DNA
sequences.

4. WHY SEQUENCING A GENOME?
One of the interesting things about biological organisms is their
remarkable similarity at the molecular level, despite their obvious
outward differences. For instance, many genes are found in
morphologically different organisms despite the phylogenetic distance
between them. Not only are these genes very similar in their DNA
sequence composition; they also tend to perform the same functions.
Thus, by understanding the function of a gene in one organism,
scientists can get an idea of what function that gene may perform in a
more complex organism such as humans.

5. SHOTGUN SEQUENCING
 Sequencing large amounts of genetic information in a reasonable
amount of time has only recently become possible, using a "shotgun"
approach where a genome is fragmented into millions of short pieces
that are sequenced simultaneously and then reassembled all at once.
 The term "shotgun" to denote sequencing of a large number of
random DNA clones was first coined by F. Sanger and co-workers in
1982. They used this method to sequence the entire genome of
bacteriophage λ (lambda), a well studied virus

6. PRINCIPLE
The key to shotgun sequencing is that many of the fragments contain
overlapping sequences or segments called contigs (short for
contiguous sequences). Once the fragments have been sequenced,
the data can be assembled on a computer to reconstruct the original
sequence of the entire chromosome.

7. STEPS IN SHOTGUN SEQUENCING
1. RANDOM PHASE
a. Fragmentation
b. Cloning
c. Sequencing

8. 2. ASSEMBLY PHASE
3. FINISHING PHASE

9. STRATEGIES FOR SHOTGUN SEQUENCING
HIERARCHICAL SHOTGUN
SEQUENCING
This method is also known as
BAC-to-BAC method, employed by
the DOE and NIH funded HGP, is
slow because it depends on
mapping the genome to be
sequenced and obtaining sets of
partially ordered, overlapping
BACs. Also referred to as the map-
based method, it was developed
from procedures used in individual
labs in the late 1980s and 90s.

10. STEPS IN HIERARCHICAL SHOTGUN SEQUENCING

13. Advantages and Disadvantages
 The advantage to the hierarchical approach is sequencers are less
likely to make mistakes when assembling the shotgun fragments into
contigs (as long as full chromosomes). The reason is that the
chromosomal location for each BAC is known, and there are fewer
random pieces to assemble.
 The disadvantage is the slow process of sub-cloning and mapping of
the source clones, requiring significant human manipulation.

14. WHOLE GENOME SHOTGUN SEQUENCING
Whole genome shotgun sequencing is a much faster approach, and
enabled researchers to speed up the timetable for sequencing
enormously. The shotgun method was developed by J. Craig Venter
and his associates in 1996 when he was at the Institute for Genomic
Research (TIGR).

15. STEPS IN WHOLE GENOME SHOTGUN SEQUENCING

16. Advantages and Disadvantages
This shotgun method is faster and less expensive, but it is more
prone to errors due to incorrect assembly of finished sequence.

17. Which method is better?
It depends on the size and complexity of the genome. With the human
genome, each group believes its approach to be superior to the other.
We only have draft sequences and each has gaps and unfinished
regions so it is not possible to say for sure. It is worth mentioning that
Celera had access to the HGP data but the HGP did not have access
to the Celera data. Furthermore, since the Celera data is not freely
available, most investigators will use the HGP sequence for further
research. Therefore, we may never know which method "won".

18. Milestones in Genome Sequencing
 First Cellular Organisms
The first bacterial genome, Haemophilus influenzae Rd 1.83 Mbp (Fleischmann
et al. , 1995), sequenced using the whole genome shotgun sequencing approach.
First eukaryotic organism Saccharomyces cerevisiae 13Mbp (Goffeau et
al..,1996).
A bacterium, Escherichia coli 4.60Mbp (Blattner et al. , 1997) is a preferred model
in genetics, molecular biology, and biotechnology..
 First Multicellular Organisms
A small invertebrate, the nematode or roundworm, Caenorhabditis elegans 97
Mbp (C. Elegans Sequencing Consortium, 1998). A fruit fly, Drosophila
melanogaster 137 Mb (Adams et al., 2000).
Homo sapiens 2.9Bbp International Human Genome Sequencing Consortium,
Nature, Feb.15, 2001 and Venter et al. Science Feb. 16, 2001.
Mus Musculus 2.6Bbp Celera Genomics, spring 2001, unpublished. According to
Genomes Online Database (Bernal et al. , 2001), on 10th of April, 2002, the total
of 84 complete genomes have been published, 271 ongoing sequencing projects
of prokaryote genomes and 178 eukaryotic sequencing projects.

19. Applications of Shotgun Sequencing
 Medicine, including the diagnosis and early detection of genetically
inherited diseases; development of gene therapy and custom-fit
drugs (pharmacogenomics)
 Alternative Energy sources, including the development of biofuels
utilizing photosynthetic and microbial systems, gleaned from the
study of bacterial genomics
 Evolutionary studies, including lineage studies and phylogenetics
 DNA Forensics, involving the use of DNA to identify individuals for
crime tracking.

20. CONCLUSION
Shotgun sequencing is a laboratory technique for determining the DNA
sequence of an organism's genome. The advent of shotgun sequencing
techniques advanced the initial mapping of the human genome by
years, and it has provided biologists, geneticists, and doctors with a
powerful new tool. The ability to sequence genetic data rapidly has
potential benefits not only for life and health science professionals, but
also for the public at large.

21. REFERENCES
The Principles of Shotgun Sequencing and Automated Fragment
Assembly
C.Martti T. Tammi
martti.tammi@cgb.ki.se
Center for Genomics and Bioinformatics, Karolinska Institute, Sto ckholm,
Sweden April 13, 2003
Shotgun sequencing of Human genome
J. Craig Venter; Mark D. Adams; Granger G. Sutton; Anthony R. Kerlavage;
Helminton O. Smith
Science, New series, Vol. 280, No. 5369 (June 5, 1998) 1540-1542