Genome sequencing is the process of determining the order of nucleotide bases - A, C, G, and T - that make up an organism's DNA. Shotgun sequencing involves randomly breaking the genome into small fragments, sequencing those pieces, and reassembling the sequence by identifying overlapping regions. It was originally used by Sanger to sequence small genomes like viruses and bacteria. There are two main methods - hierarchical shotgun sequencing for larger genomes containing repeats, and whole genome shotgun sequencing for smaller genomes.

1. Shotgun Genome
Sequencing
Saif Saleh Mohsen Ali
Dept of Biochemistry
Genetics & Genomics

2. • Genome is all genetic material of an organism. It consists of DNA (or RNA in
RNA viruses). The genome includes both the genes (the coding regions) and the
noncoding DNA, as well as mitochondrial DNA and chloroplast DNA. The study
of the genome is called genomics.
• What is the genome sequencing?
• Genome sequencing is the technique that allows researchers to read the
genetic information found in the DNA of anything from bacteria to plants to
animals
• Sequencing involves determining the order of bases, the nucleotide subunits-
Adenine(A), Guanine(G), Cytosine(C) and Thymine(T), found in DNA
What is the genome

3. Determining the Sequence of DNA
Methods:
1) Maxam and Gilbert chemical degradation method.
2) Chain termination or Dideoxy method • Fredrick Sanger .
3) Genome sequencing method:
• Shotgun sequencing
• Clone contig approach
4) Second generation sequencing methods:
• Pyrosequencing
• Nanopore sequencing
• Illumina sequencing
• Solid sequencing

4. Factors that determine sequencing strategy are :-
• a) Genome size
• b) Chromosomal structure
• c) Repeat content and character
• d) Desired end product

5. What is shotgun sequencing?
• Shotgun sequencing involves randomly breaking up DNA sequences
into lots of small pieces and then reassembling the sequence by
looking for regions of overlap.
• Shotgun sequencing was originally used by Fred Sanger and his
colleagues to sequence small genomes such as those
of viruses and bacteria .
• In whole genome shotgun sequencing the entire genome is broken
up into small fragments of DNA for sequencing.

6. Shotgun Genome Sequencing
•Two different methods used :-
1) Hierarchical shotgun sequencing
Useful for sequencing genomes of higher vertebrates
that contain
repetitive sequences
2) Whole genome shotgun sequencing
Useful for smaller genome

8. History
• Shotgun sequencing has been developed for sequencing
of large fragments of DNA in 1979.
• The first genome sequenced by shotgun sequencing was
that of cauliflower mosaic virus, published in 1981.
• Shotgun sequencing was the initiative for full genome
sequencing.

9. The principal of shotgun sequencing
• The chain termination method of DNA sequencing can only
be used for fairly short strands of 100 to 1000 base pairs.
•Longer sequences are subdivided into smaller fragments that
can be sequenced separately, and subsequently they are re-
assembled to give the overall sequence.

10. Steps of shotgun genome sequencing
• Break genome into smaller fragments.
• Sequence those smaller pieces.
• Piece the sequences of the short fragments together .

12. Example for Shotgun Sequencing

13. Advantages of shotgun sequencing:
• By removing the mapping stages, whole genome shotgun sequencing is
a much faster process than clone-by-clone sequencing.
• Whole genome shotgun sequencing uses a fraction of the DNA that
clone-by-clone sequencing needs.
• Whole genome shotgun sequencing is particularly efficient if there is an
existing reference sequence.
• It is much easier to assemble the genome sequence by aligning it to an
existing reference genome .
• Shotgun sequencing is much faster and less expensive than methods
requiring a genetic map.

14. Disadvantages of shotgun sequencing:
• Vast amounts of computing power and sophisticated software are
required to assemble shotgun sequences together. To sequence the
genome from a mammal (billions of bases long), you need about 60
million individual DNA sequence reads.
• Errors in assembly are more likely to be made because a genetic map
is not used. However these errors are generally easier to resolve than
in other methods and minimised if a reference genome can be used.
• Whole genome shotgun sequencing can only really be carried out if a
reference genome is already available, otherwise assembly is very
difficult without an existing genome to match it to.
• Whole genome shotgun sequencing can also lead to errors which need
to be resolved by other, more labour-intensive types of sequencing,
such as clone-by-clone sequencing.
• Repetitive genomes and sequences can be more difficult to assemble.
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