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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
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IOP Publishing
Introduction to Pharmaceutical Biotechnology, Volume 2
(Second Edition)
Enzymes, proteins and bioinformatics
Ahmed Al-Harrasi, Saurabh Bhatia and Ajmal Khan
Chapter 7
Introduction to genomics
7.1 Introduction
In 1910 several researchers first worked together to identify and map genes in
organisms of interest. From then, efforts progressively focused on specific organisms, e.g. maize, mice, Drosophila, bacteria and yeast.
Genetic mapping involves three important steps:
• Identification of spontaneous mutations, or mutants induced by chemical or
physical agents or identifying the locus accountable for variation.
• Sequencing the region in cases and controls to define causal mutation(s).
• Investigating the molecular and cellular variations causes and changes in the
functions of the genes discovered.
For spontaneous mutations, DNA sequence analysis has revelaed the types of
mutations that are responsible for several hereditary diseases. Many disorders are
due to deletions or duplications, including repeated sequences. Multiple sources,
such as errors in DNA replication, lesions, and transposable genetics and elements
are responsible for spontaneous mutations. When this type of mutation exists it can
be used for linkage studies and planning for linkage maps. It was observed by Drake
et al in 1998 that the rate of occurrence of spontaneous mutation per genome is
extraordinarily identical within extensive groups of organisms, but varies extremely
among groups [1]. Physical mappings of genes in organisms such as Drosophila were
also created. This strategy was effective and is extensively used in genetics. However,
one of the major shortcomings of this method is that at least one mutation for the
respective gene in the genome is essential. Attaining mutations of each gene is very
difficult and also labor intensive. Moreover, the genetic mutation may also impact
phenotype. Mutations frequently have a harmful outcome, making it practically
impossible to map the mutated gene. In the mid-1980s, researchers began using
doi:10.1088/978-0-7503-5387-8ch7 7-1 ª IOP Publishing Ltd 2024. All rights,
including for text and data mining (TDM), artificial intelligence (AI) training, and similar technologies, are reserved.
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
recombinant DNA technology for investigation. In this method, a number of clones,
called a genomic library, are established. These are paired together into overlapping
sets and brought together they provide genetic material and physical maps for the
complete genome. The clones are initially sequenced and thereafter all the genes in
the genome are identified from this sequence.
7.2 Characterizations in genomics
In 1987, the word genomics was coined by the geneticist Thomas H Roderick, at the
Jackson Laboratory, Bar Harbor, ME [2]. During that period, the term was
considered to mean mapping and sequencing to examine the arrangement and
organization of genomes. Currently, genomics comprises the sequencing of
genomes, determination of the complete set of proteins encoded by an organism,
and the functioning of genes and metabolic pathways in an organism. Thus
genomics not only deals with the determination of the genetic information present
in an organism, but also with understanding the mechanism by which this
information is used by the organism. The word genome was presented by
H Winkler in 1920. He used the word genome for the complete set of chromosomal
and extra chromosomal genes found in an organism, including viruses. This term is
used in a similar sense in modern genetics [3].
Vast amounts of information are available in genomics. Understanding and
management of this amount of information requires advanced computers and
specific software systems. Bioinformatics is a developing area which involves
development and application of computer hardware and software to the procurement, storage, analysis and imaging of biological information. Databanks, which
are necessary for the storage and examination of genetic information, have now
become important tools for geneticists. Genomics also involves the investigation of
the products encoded by genetic material, including the number of genes that are
expressed, their period of expression, the kind and level of any post-translational
modification of the gene product, the role of the encoded protein, and its site in
various cellular compartments.
The field of genomics is divided into the following two areas:
• Structural genomics.
• Functional genomics.
In structural genomics the complete sequence of genetic material or the whole set of
proteins produced by an organism is determined by different techniques. This can be
achieved using the following steps:
• Assembling of high resolution genetic and physical maps.
• Genome sequencing.
• Evaluation of the whole set of proteins produced in an organism. Analysis is
required to determine the protein’s three-dimensional structure.
Functional genomics deals with the study of the functioning of genes and metabolic
pathways. This means the patterns of genetic expression in an organism.
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
7.3 Historical background
In 1984, the concept behind genome sequencing was first presented in the scientific
community. Later, in 1986, a scheme was set for sequencing of the human genome.
The Human Genome Project was announced to the public on 1 October 1990 [4].
The aim of the genome projects is to collect information on the structure of DNA in
human chromosomes and those of other organisms. The next objective is to develop
novel skills to accomplish mapping and sequencing. European scientific communities explored a number of genome projects on yeast, bacteria, Drosophila and
Arabidopsis thaliana in 1988, and in 1990 started a new two year program on the
human genome [5]. In 1995, the genetic material of the gram-negative bacterium,
Haemophilus influenza (which is naturally transformable), was the first to be
sequenced. In 1997, the complete genetic material of Escherichia coli (a 4 639
221-base pair sequence) was sequenced [6]. Among eukaryotic organisms, genetic
materials of yeast (Saccharomyces cerevisiae) and a worm (Caenorhabditis elegans)
were the first to be sequenced in 1999. One year after this breakthrough, Drosophila
melanogaster and A. thaliana were sequenced [6]. Finally, on 26 June 2001, a rough
draft of the human genome was published.
Separate drafts were achieved by the public-funded Human Genome Sequencing
Consortium and the private company Celera Genomics, established by Craig
Venter. However, the draft sequence was published collaboratively after involvement by the US President.
7.4 Genome sequencing
Defining the arrangement of nucleic acid residues in living specimens is an essential
part of an extensive range of research applications. A simplified procedure for the
sequencing of the whole genome is depicted in figure 7.1. For almost 50 years,
investigators have explored various techniques to sequence DNA and RNA
molecules, which involve sequencing short oligonucleotides to millions of bases,
interpretation of the coding sequence of a single gene, and rapid and extensively
available complete genome sequencing [7]. Genome sequencing is a highly complicated and challenging procedure. In one attempt, a portion of 500–600 bp (at best,
1000 bp) can be sequenced easily, however, most genomes are exceptionally large
such as 4.2 × 10
sequence of a genome has to be acquired in a particularly large number of small
fragments. These small fragments then have to be assembled into a sequence for the
genome. For sequencing, fragments are synthesized by splicing the genomic DNA
into fragments at different locations. Consequently the actual site of the fragment in
the genome has to be experimentally evaluated. In addition, the fragments
subsequently produced typically overlap other fragments at their ends. The derived
fragments from an organism’s genetic material are cloned in a desirable vector.
Ultimately this produces a genomic library of the organism.
For sequencing, cloning of the fragments is necessary to generate a large number
of copies of each fragment required. The following two methods are available for the
sequencing of genomes:
6
bp for E. coli and 3.2 × 109for humans. Thus for sequencing, the
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Figure 7.1. Sequencing of a complete genome.
• Shotgun sequencing.
• Clone-by-clone sequencing.
In both of these approaches, the principal step is to produce a genomic library for
a particular chromosome.
7.4.1 Clone-by-clone sequencing
The basis of shotgun and clone-by-clone genome sequencing is the same, however,
the difference iss that in clone-by-clone sequencing researchers create a DNA library
of the pieces of DNA clones obtained from the sequence that was used in the first
place. Data management and the mounting of DNA contigs (overlapping sequences)
is a lot easier computationally speaking. In shotgun sequencing the same end is
achieved but without cloning and libraries. Researchers directly mount contigs from
sequenced genome pieces, therefore better computers are required. Shotgun strictly
means the sequencing based on the random shearing and consecutive construction of
contigs.
During clone-by-clone sequencing the genome or considered genetic material is
fragmented or broken into large pieces (150 kb). The actual position of these
fragments in the chromosomes is mapped to support assembling them after
sequencing. The fragments are first aligned into contigs (overlapping sequence
data, i.e., a cluster of sequences which is derived after the completion of the
sequencing process, which eventually provides the sequence of a single fragment).
These contigs form a physical map of the genome which is eventually used to guide
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
sequencing and assembly. It is also called directed sequencing of BAC contigs.
During the initial procedure, BAC clones (functional fertility plasmids) are
introduced to the contigs and these a.e. then further introduced into a bacterial
cell for growth. The fragments of DNA under consideration are copied each time the
bacteria multiply to synthesize multiple duplicate copies. A BAC clone (80–100 kb)
has a long DNA fragment duplicated into it. This piece is then further employed to
produce cosmid clones and plasmid clones. These synthesized clones have progressively smaller DNA fragments. Now each individual chunk is 500 bp stretched;
a more suitable size for sequencing. These synthesized fragments are incorporated
into a vector that has a well-known DNA sequence. Then DNA fragments are
sequenced, starting with an identified sequence of the vector and extending out into
the unidentified sequence of the DNA. Following this type of sequencing even small
chunks of DNA are joined together by exploring available overlapping locations to
assemble the bulky chunks that were initially incorporated into the BACs. This
assembly is done by processors (computers) which identify locations of overlap and
assemble the DNA sequence. Using the map created during the initial phase, the
large pieces can be arranged again into chromosomes as part of the whole genome
sequence. This method was introduced during the 1980s and 1990s to initially
sequence the genetic material of the C. elegans (the nematode worm) and
S. cerevisiae (yeast). During the sequencing of Human Genome Project (2001),
clone-by-clone sequencing was favored. There are a number of advantages to
clone-by-clone sequencing: (1) each fragment of DNA is taken from a well-known
region of the genome, so it is comparatively easy to regulate whether there are any
gaps in the sequence; (2) as a genome map is followed this method offers more
reliable assembly, so researchers know where larger pieces/fragments belong in
relation to each other; and (3) as each and every piece of DNA is different, numerous
people can work on the genome simultaneously.
7.4.2 Human whole-genome shotgun sequencing
The determination of the whole sequence of genes in a genome is required to
understand the genetic basis of an organism. The sequencing of large numbers of
gene fragments and even complete genomes is only possible with the development of
tools for DNA sequencing. Novel significant tools such as physical mapping, DNA
sequencing and sequence analysis have been established. To increase the output,
robotic processes for sample preparation and novel software for sequence analysis
have been applied [8]. A simplified representation of human whole-genome shotgun
sequencing is illustrated in figure 7.2.
In 1997, Weber and Myers published a study entitled ‘Human whole-genome
shotgun sequencing’. They contended, by means of computational models, that in
the place of long reads, short reads would cover sufficient data to sequence the first
human genome, which would not only be economical, but quicker than the Human
Genome Project. This was so fiercely debated that Phillip Green published a review,
describing why shotgun sequencing was not possible. Both studies were finally
accepted and published side by side in the journal Genome Research.
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Figure 7.2. Human whole-genome shotgun sequencing.
Shotgun sequencing includes arbitrarily splicing of DNA sequences into numerous small fragments and then reconstructing the sequence by examining for regions
of overlap. Because of their size and structural sophistication, large, mammalian
genomes are the most problematic to clone, sequence and assemble [9].
For such genomes, the method known as clone-by-clone sequencing, although
reliable and systematic, takes a very long time. With the development of cheaper
sequencing techniques and more advanced computer databases, geneticists can thus
rely on whole-genome shotgun sequencing to deal with large, complex genomes.
Fred Sanger and co-workers initially used shotgun sequencing to sequence small
genomes, for example, the genomes of viruses and bacteria. Complete genetic
material shotgun sequencing avoids the long mapping and cloning steps that make
clone-by-clone sequencing so gradual. During this procedure the complete genome is
fragmented into small pieces of DNA for sequencing. These pieces are often of
variable sizes, extending from 2–20 kb (2000–20 000 bp) to 200–300 kb (200 000–
300 000 bp). Then these pieces are sequenced to examine the order and arrangement
of the DNA bases (A, C, G and T). Subsequently, the sequenced fragments are
collected together by computer databases that determine where fragments overlap.
In analogy, this technique is akin to chopping several copies of a book (which is here
the genome or genetic material), blending up all the pieces and at that point
reuniting the original text (genome/genetic material) by finding fragments/pieces of
DNA with text that overlaps and piecing the book back together based on these.
This technique was used by Venter (founder of Celera Genomics) to sequence the
human genome. Venter desired to sequence the human genome quicker than the
reported goal, and considered shotgun sequencing to be the best approach. For
assembly, Venter initially used the reported clone-by-clone data from the Human
Genome Project. Currently, with advancements in computational technology, entire
genome shotgun sequencing is being employed to further advance the accuracy of
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
present genome sequences, for example, the reference human genome. This
procedure can be utilized to eliminate errors, fill in gaps or rectify parts of the
sequence that were initially assembled inaccurately (by clone-by-clone sequencing).
Consequently, the reference human genome is continuously being developed to
guarantee that the genome sequence is of the utmost high standard. There are
number of merits to shotgun sequencing, such as:
• By eliminating the mapping steps, entire genome shotgun sequencing (EGSS)
is a much quicker process than clone-by-clone sequencing.
• EGSS utilizes a piece of the DNA that clone-by-clone sequencing needs.
• EGSS is mainly effective if there is any available reference sequence against
an unidentified genome. It is much more convenient to assemble the genome
sequence by assembling it to a present reference genome.
• Shotgun sequencing is much quicker and economical than the procedures
employed for genetic mapping.
However, there are still various disadvantages to shotgun sequencing, such as:
• An enormous amount of computing power and advanced and sophisticated
software are necessary to align shotgun sequences together.
• For sequencing the genomic material of a mammal (billions of bases long),
60 million individual DNA sequence reads are required.
• Since a genetic map is not used, the possibility of error in assembly is greater.
However, comparatively these errors are usually easier to resolve than in
other procedures and are minimized if a reference genome can be used.
• EGSS assembly is very challenging without an existing reference genome to
match it to.
• EGSS can also result in errors which must be determined by other, more
labor-intensive types of sequencing, such as clone-by-clone sequencing.
7.4.3 Compilation of genome resources
Four main objectives were set out for the Genome Sequence Compilation [10]:
• To determine evolutionarily conserved sequence motifs, mainly outside
protein-coding genes, which are accountable for regulatory and other critical
genomic functions.
• To explore new models of human disease and heritable phenotypes.
• To offer a starting point for the evaluation of the expansion, contraction, and
adaptation of gene families in different evolutionary lineages.
• To offer a outline for the reconstruction of genome organization, content and
dynamics that have happened through the mammalian radiations.
Genome sequencing projects required the production of high throughput tools
that produce desirable information quickly. This can be achieved by the employment of advance computers to manage this vast store of information. These
computational tools have given birth to a new area called bioinformatics. Manual
manipulation of gene data is time consuming, but bioinformatics can deal with the
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