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Genome Sequencing and Assembly

The document discusses genome sequencing and assembly. It provides background on several genome projects including the Human Genome Project, Neanderthal Genome Project, and 1000 Genomes Project. It then describes different sequencing methods such as Sanger sequencing and Pacific Biosciences sequencing. Finally, it discusses next-generation sequencing technologies and how they have revolutionized genomic research by allowing faster and cheaper whole genome sequencing compared to previous methods.

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106 views

Genome Sequencing and Assembly

The document discusses genome sequencing and assembly. It provides background on several genome projects including the Human Genome Project, Neanderthal Genome Project, and 1000 Genomes Project. It then describes different sequencing methods such as Sanger sequencing and Pacific Biosciences sequencing. Finally, it discusses next-generation sequencing technologies and how they have revolutionized genomic research by allowing faster and cheaper whole genome sequencing compared to previous methods.

Uploaded by

muazzam22
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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Name: Syed Muazzam Ali Shah

Student Id: s1069108


Course Name: CB535 Applications of Bioinformatics
Seminar Title: Genome Sequencing and Assembly

The Human Genome Project (HGP) was an international scientific research project and
world largest collaborative biological project, the basic aim of this project to identifying
the sequence of nucleotide base pairs involve in the formation of human DNA. It is also
responsible for determining and mapping all type of genes related to human genome
from both physical and functional standpoint. This project launched in 1990, completed
in April 2003, and funded by the US government.

In 2001, the Human Genome Project (HGP) announced the first draft and initial
investigation about human genome sequence. This draft contained more than 90% of
the human genome. In this initial draft, the more amazing thing is that the estimated
number of genes was lower than the expected number of genes.

The Neanderthal genome project founded in July 2006 is a project developed by a team
of scientist to sequence the Neanderthal genome. The project released their initial draft
in May 2010.

The 1000 Genomes Project released in January 2008. It is the first project related to the
sequencing of large number of human genomes. It also provide a complete public
catalog for human genetic variation.

DNA sequencing is the procedure of determining the sequence of nucleotide bases (As,
Ts, Cs and Gs) in a portion of DNA. Sequencing a whole genome is a complicated task.
It involves fragmenting the DNA of the genome into numerous shorter fragments,
sequencing the fragments and assembling the sequences into a single long consensus.
However with the advent new approaches over the past two decades, genome
sequencing is now much faster and less costly than it was during the Human Genome
Project.
Sanger sequencing also known as “chain termination method” is a method for
identifying the nucleotide sequences of DNA. This method was developed by Frederick
Sanger and his colleagues in 1977, therefore the name the Sanger Sequence.
There are three main steps involved in Sanger sequencing, which are explained below;
1. The first step involve generating n DNA fragments of variable lengths, each ended
with a tagged nucleotide, where n is the number of nucleotide bases in the target DNA
sequence. This is achieved by merging DNA primer, nucleotides (dATP, dCTP, dGTP,
dTTP), DNA polymerase, the DNA sequence of interest, and marked
dideoxynucleotides (ddATP, ddCTP, ddGTP, ddTTP). No nucleotide can be
supplemented to the DNA chain once a dideoxynucleotides has been incorporated,
hence each fragment will end with a labelled nucleotide.
2. While in the second step, n DNA sequences are detached with respect to the length
using capillary gel electrophoresis. The shorter fragments move faster as compared to
the lengthier fragments. The conclusion is that the DNA pieces are passed into the third
step from shortest to longest sequence.
3. Finally in the third step a laser excites the label on the nucleotide at the end of each
sequence. Each base is marked with a different label, so the light produced by each
excited nucleotide can be tied to the correct base. The laser produces a chromatogram
presenting the fluorescent peak of each nucleotide. The chromatogram has the
nucleotides in the correct order because of the electrophoresis.

Pacific bioscience sequencing make use of Single Molecule Real Time (SMRT)
technology, which empowers the real time identification of nucleotide incorporation
events throughout the elongation of the replicated strand from non-amplified single
stranded template. SMRT technology uses nucleotides having a fluorescent tag on the
phosphate chain of the nucleotide rather than on the base. Therefore, incorporated
nucleotides are identified based on the related fluorophore that is released and
dissipated upon cleavage of the phosphate chain.
Some of the advantages of SMRT sequencing are given below;
 Longest average read lengths.
 Highest consensus accuracy.
 Uniform coverage.
 Simultaneous epigenetic characterization.
 Single-molecule resolution.
Next generation sequencing (NGS), massively parallel or deep sequencing are the
analogous terms. It characterize DNA sequencing technology, which has
revolutionized genomic research. With the help of NGS, a complete human genome
can easily be sequenced with in only a single day. It is much faster as compare to the
previous Sanger sequencing technology, which is used to decipher the human genome,
needed more than a decade to produce the ultimate draft.
Next generation sequencing allow researchers to:
 Quickly sequence complete genomes.
 Focus deep in sequence target regions.
 It helps to discover novel RNA variants and splice sites by using RNA
sequencing.
 Analyze epigenetic factors like DNA- protein interactions and genome wide
DNA methylation.
 Study microbial diversity in humans or in the environment.
 It also play role in sequencing cancer samples to study rare somatic variants,
tumor clones and many more.
There are a variety of next-generation sequencing techniques that use different
technologies. However, most share a common set of features that distinguish them from
Sanger sequencing:
Highly parallel: Many sequencing reactions take place at the same time.
Micro scale: Reactions are tiny and many can be done at once on a chip.
Fast: Because reactions are done in parallel, results are ready much faster.
Low-cost: Sequencing a genome is cheaper than with Sanger sequencing.
Shorter length: Reads typically range from 505050 -700700700 nucleotides in length.

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