Genome sequencing involves determining the complete DNA sequence of an organism's genome, which includes the position of genes, regulatory regions, and coding sequences. Shotgun sequencing, where DNA is randomly fragmented and reassembled, is a key method for sequencing genomes, particularly useful for larger genomes and ancient DNA studies. Various sequencing technologies, including Sanger and next-generation sequencing, are employed for this purpose, and genome assembly requires specialized computational techniques to accurately reconstruct the original genome.

1. Genome Sequencing, Shot
gun Sequencing

2. Genome: Complete set of genetic information from
a haploid set of chromosomes of an organism.
• Genome is found in every cell, inside the
nucleus.
Genomics: The study of structure and function of
whole genomes and their interactions rather than
single genes or proteins.
INTRODUCTION

3. The sequence information of genome will show
• The position of every gene along the chromosome
• The regulatory regions that flank each gene
• The coding sequence that determines the protein produced by each gene
Genome sequencing is the process of determining the complete DNA
sequence of an organism's genome.
Sequencing involves determining the order of bases.
Importance: Essential for understanding genetic makeup, evolution, and
disease.

4. Collecting a sample containing the DNA of interest. This
could be a blood sample, tissue biopsy, saliva, or any
other biological material depending on the organism
being studied.
DNA Shearing
Sample Collection: DNA Extraction:
Scientists cut the DNA into short fragments of known
length, either by using enzymes or mechanical
disruption
DNA Library Preparation
Scientists make many copies of each DNA fragment
using a process called polymerase chain reaction
(PCR)
This is typically done using various biochemical
methods that break open the cells and separate the
DNA from other cellular components such as proteins
and lipids.
Basic steps in genome sequencing

5. Basic steps in genome sequencing
Sequencing
The prepared DNA fragments are then loaded
onto a sequencing platform where they undergo a
series of biochemical reactions to determine their
nucleotide sequence. There are several sequencing
technologies available, including Sanger
sequencing, Illumina sequencing
Genome Annotation
After the genome sequence is assembled, it needs
to be annotated to other functional elements
within the genome. This involves predicting gene
locations, identifying coding and non-coding
regions such as promoters, enhancers, and
repetitive sequences.
DNA Sequence Analysis
The sequencer produces millions of DNA reads, and
specialized computer programs are used to put them
together in the correct order.
Validation
This can be done using various experimental
techniques such as polymerase chain reaction
(PCR), Sanger sequencing, and functional
assays to confirm the presence and
functionality of predicted genes and regulatory
elements.

7. Determining the sequence of DNA
Methods
• Maxam and Gilbert chemical degradation method
• Chain termination or deoxy method Frederick Sanger method
Genome sequencing method:
• Shotgun sequencing
Second generation sequencing methods:
• Pyro sequencing
• Nanopore sequencing
• Illumina sequencing

8. Factors determining sequencing strategy
• Genome size
• Chromosomal structure
• Repeat condensed character
• Desired end product

9. Shotgun sequencing
• It is a method used in molecular biology to determine the DNA sequence
of an organism's genome.
• It is called "shotgun" sequencing because it involves randomly breaking
up DNA sequences into lots of small pieces and then reassembling the
sequence by looking for regions of overlap.
• Originally used by Sanger and his colleagues to sequence small genomes
such as those of viruses and bacteria.

10. History
1970s: Early DNA Sequencing Techniques (Frederick Sanger)
1980s: Initial Concepts of Shotgun Sequencing
1990s: Shotgun Sequencing of Small Genomes (Bacteria, Virus)
Notable achievements include the sequencing of the bacterium
Haemophilus influenzae in 1995, the first complete genome sequence of
a free-living organism.
2000s: Human Genome Project and Next-Generation Sequencing
2005: Rapid Advances in NGS Technologies

11. Principle of shotgun sequencing
• The chain termination method of DNA
sequencing can only be used for fairly short
strands of 100 to 1000 base pairs.
• The principle of shotgun sequencing involves
breaking the genome into small, random
fragments, sequencing these fragments, and
then assembling them back together to
reconstruct the original genome sequence.

12. TWO METHODS IN GENOME SEQUENCING
Heirarchial shot gun
sequencing
Useful for sequencing genomes
of higher vertebrates that contain
repetitive sequences
Whole genome
sequencing
It is useful for smaller
genome

13. Hierarchical shotgun
sequencing
Imagine a jigsaw puzzle with
billions of tiny pieces, that's the
challenge of sequencing DNA.
Hierarchical shotgun sequencing
breaks down this complexity by
breaking DNA into manageable
fragments

14. Hierarchical genome sequencing
It is a method of DNA sequencing that
involves breaking down the genome into
smaller, more manageable fragments,
sequencing those fragments, and then
assembling the sequence data to
reconstruct the entire genome in a
hierarchical manner.
Principle
• Break the genome into manageable
fragments.
• Sequence each fragment.
• Assemble the sequence data to
reconstruct the genome.

15. The genome is initially
fragmented into manageable
pieces using physical or
enzymatic methods.
Fragments are cloned into
vectors, such as bacterial
artificial chromosomes
(BACs) or yeast artificial
chromosomes (YACs), to
generate larger insert clones.
Cloning helps in handling
larger DNA fragments that
are difficult to sequence
directly.
The cloned fragments are further
subcloned into smaller inserts to
facilitate sequencing. This step
generates subclones with insert sizes
suitable for sequencing
Fragmentation
Cloning
Subcloning
Steps

16. Steps
Shotgun Sequencing
Each subclone is sequenced individually
using high-throughput sequencing
technologies, such as Sanger sequencing
or next-generation sequencing platforms.
This step generates sequence reads for
each subclone.
Integration
Contigs are integrated to
reconstruct the entire genome
sequence. This integration
involves merging overlapping
contigs and filling gaps between
them to generate a
comprehensive genome
assembly
Assembly
Computational algorithms are used
to assemble the sequence reads from
individual subclones into contiguous
sequences called contigs. Contigs
represent larger portions of the
genome and are assembled
hierarchically based on overlapping
sequences from subclones.

17. Advantages and Disadvantages
• Allows for sequencing larger genomes by
breaking them into manageable, larger
pieces initially.
• Reduces the complexity of sequence
assembly by sequencing and assembling
larger fragments first.
• Can be more cost-effective for sequencing
large genomes compared to whole genome
shotgun sequencing.
Advantages
• Requires additional steps for fragmenting
and sequencing larger DNA segments,
which can increase the overall time and
cost of sequencing.
• May miss smaller genetic variations or
sequences present within the larger
fragments if not adequately covered
during sequencing
Disadvantages

19. Whole genome Sequencing
• In this approach, the genome is decomposed
directly into individual random reads.
• Then attempts to assemble the genome as a
whole.
Principle:
• Randomly fragment the genomic DNA into
small, overlapping fragments.
• Sequence the fragments using high-throughput
sequencing technologies.
• Assemble the sequence reads into contiguous
sequences using computational algorithms.

20. Steps
• Isolation of Genomic DNA: The process begins with the isolation of
genomic DNA from the organism of interest.
• Random Fragmentation of Genomic DNA: The isolated genomic DNA is
then randomly fragmented into small pieces. This can be achieved through
methods such as sonication or nebulization.
• Size Selection Using Electrophoresis: The fragmented DNA is subjected
to size selection using electrophoresis, which allows for the isolation of
DNA fragments of the desired size range.

21. Steps
• Library Construction: This involves the ligation of sequencing adapters to
the DNA fragments, which are essential for the subsequent steps of the
sequencing process.
• Paired-End Sequencing (PE Sequencing): The DNA library is subjected to
paired-end sequencing, which involves sequencing both ends of the DNA
fragments. This step provides crucial information about the relative positions
and orientations of the DNA fragments within the genome.
• Genome Assembly: This process involves using specialized computer
programs to align and merge the individual DNA reads to reconstruct the
original genome sequence. The overlapping sequences are used to piece
together the complete genome.

23. Advantages
• Provides a comprehensive and unbiased view
of the entire genome, including smaller genetic
variations and sequences.
• Can be more suitable for sequencing smaller
genomes or genomes with less repetitive regions.
• Offers flexibility in sample preparation and
sequencing, allowing for faster turnaround times
and lower costs compared to hierarchical
shotgun sequencing
Disadvantages
• Requires more computational
resources and sophisticated
algorithms for assembling the
fragmented sequences accurately.
• May encounter challenges in
assembling repetitive regions or
resolving complex genomic
structures.

25. Applications
• Large Genome sequencing : Used in sequencing large and complex genomes.
Example: Sequencing of maize genome , Human genome project
• Metagenomics: Used to study microbial communities in environmental sample
Example: Global ocean sampling expedition.
• Cancer Genomics: Used to identify genetic mutations and alterations in cancer cells
Example: The Cancer Genome Atlas project
• Ancient DNA Studies: Shotgun sequencing has been crucial in studying ancient DNA from
archaeological samples.

26. References
• Ranjan, R., Rani, A., Metwally, A., McGee, H. S., & Perkins, D. L. (2016). Analysis
of the microbiome: Advantages of whole genome shotgun versus 16S amplicon
sequencing. Biochemical and Biophysical Research Communications, 469(4), 967–
977.
• Marra, M. (2024). Whole genome sequencing, genetics. The Editors of Encyclopaedia
Britannica.
• Waterston, R. H., Lander, E. S., & Sulston, J. E. (2002). On the sequencing of the
human genome. Proceedings of the National Academy of Sciences, 99(6), 3712-3716.
• Koskinen, V. R., Emery, P. A., Creasy, D. M., & Cottrell, J. S. (2011). Hierarchical
clustering of shotgun proteomics data. Molecular & Cellular Proteomics, 10(6).
• https://www.bio.davidson.edu/courses/genomics/method/shotgun.html
• https://www.news-medical.net/life-sciences/Shotgun-Sequencing.aspx