Molecular Genetics
Genome Sequencing
What is a genome Types of genomes What is genomics How is genomics different from genetics Types of genomics Genome sequencing
Milestones in genomic sequencing Technical foundations of genomics Steps of genome sequencing DNA sequencing approaches
Hierarchical shotgun sequencing Markers used in mapping large genomes
Whole genome shotgun sequencing New technologiesGenome sequencing achievment in Bangladesh
Benefits of Genome Research
At a glance At a glance
WHAT IS A GENOME? WHAT IS A GENOME? Genome: One complete set of genetic information (total amount of DNA) from a haploid set of chromosomes of a single cell in eukaryotes, in a single chromosome in bacteria, or in the DNA or RNA of viruses.
Basic set of chromosome in a organism.
“The whole hereditary information of an organism that is encoded in the DNA”
•In cytogenetic genome means a single set of chromosomes.
•It is denoted by x. Genome depends on the number of ploidy of organism.
• In Drosophila melanogaster (2n = 2x = 8); genome x = 4.• In hexaploid Triticum aestivum (2n = 6x = 42); genome x = 7.
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The genome is found inside every cell, and in those that have nucleus, the genome is situated inside the nucleus. Specifically, it is all the DNA in an organelle.
The term genome was introduced by H. Winkler in 1920 to denote the complete set of chromosomal and extra chromosomal genes present in an organism, including a virus.
How many types of genomes are:How many types of genomes are:
1. Prokaryotic Genomes2. Eukaryotic Genomes
• Nuclear Genomes• Mitochondrial Genomes• Choloroplast Genomes
If not specified, “genome” usually refers to the nuclear genome.
• Genomics is the study of the structure and function of whole genomes.
• Genomics is the comprehensive study of whole sets of genes and their interactions rather than single genes or proteins.
• According to T.H. Roderick, genomics is the mapping and sequencing to analyze the structure and organization of genome.
WHAT IS GENOMICS?WHAT IS GENOMICS?
Origin of terminologyOrigin of terminology• The term genome was used by German botanist Hans
Winker in 1920 • Collection of genes in haploid set of chromosomes• Now it encompasses all DNA in a cell
Genomics is the sub discipline of molecular genetics devoted to the
Genomics is the sub discipline of molecular genetics devoted to the
The field includes studies of intro-genomic phenomena such as heterosis, epistasis, pleiotropy and other interactions between loci and alleles within the genome.
The sequence information of the genome will show;
The position of every gene along the chromosome, The regulatory regions that flank each gene, and The coding sequence that determines the protein
produce by each gene.
How is Genomics different from Genetics?Genetics as the study of inheritance and genomics as the
study of genomes.– Genetics looks at single genes, one at a time, like a
picture or snapshot.– Genomics looks at the big picture and examines all the
genes as an entire system.
Types of GenomicsTypes of Genomics1. Structural: It deals with the determination of the complete sequence of genomes and gene map.
This has progressed in steps as follows:
(i) construction of high resolution genetic and physical maps,
(ii) sequencing of the genome, and
(iii) determination of complete set of proteins in an organism.
2. Functional: It refers to the study of functioning of genes and their regulation and products(metabolic pathways), i.e., the gene expression patterns in organism.
3. Comparative: It compare genes from different genomes to elucidate functional and evolutional relationship.
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.
Genome sequencing is figuring out the order of DNA nucleotides.
Genome SequencingGenome Sequencing
Challenges of genome sequencingChallenges of genome sequencing
Data produce in form of short reads, which have to be assembled correctly in large contigs and chromosomes.
Short reads produced have low quality bases and vector/adaptor contaminations.
Several genome assemblers are available but we have to check the performance of them to search for best one.
Milestones in Genomic SequencingMilestones in Genomic Sequencing
1977; Fred Sanger; φX 174 bacteriophage (first sequenced genome ); 5,375 bpAmino acid sequence of phage proteinsOverlapping genes only in viruses
Fig: The genetic map of phage φX174 (Overlapping reading frames)
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1995; Craig Venter & Hamilton Smith;Haemophilus influenzae (1,830,137 bp) (1st free living).Mycoplasma genitalium (smallest free-living, 580,000 bp; 470 genes)
1996; Saccharomyces cerevisiae; (1st eukaryote) 12,068,000 bp
1997; Escherichia coli; 4,639,221 bp; Genetically more important.
1999; Human chromosome 22; 53,000,000 bp
2000; Drosophila melanogaster; 180,000,000 bp
2001; Human; Working draft; 3,200,000,000 bp
2002; Plasmodium falciparum; 23,000,000 bp
Anopheles gambiea; 278,000,000 bp
Mus musculus; 2,500,000,000 bp
2003; Human; finished sequence, 3,200,000,000 bp
2005; Oryza sativa (first cereal grain); 489,000,000 bp
2006; Populus trichocarpa (first tree) ; 485,000,000 bp
Technical foundations of genomics Molecular biology: Almost all of the
underlying techniques of genomics originated with recombinant-DNA technology.
DNA sequencing: In particular, almost all DNA sequencing is still performed using the approach pioneered by Sanger.
Library construction: Also essential to high-throughput sequencing is the ability to generate libraries of genomic clones and then cut portions of these clones and introduce them into other vectors.
PCR amplification: The use of the polymerase chain reaction (PCR) to amplify DNA, developed in the 1980s, is another technique at the core of genomics approaches.
Log
MW
Distance
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Hybridization techniques: Finally, the use of hybridization of one nucleic acid to another in order to detect and quantitate DNA and RNA (Southern blotting). This method remains the basis for genomics techniques such as microarrays.
Break genome into smaller fragments Sequence those smaller pieces Piece the sequences of the short fragments together
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 genomes
Steps of genome sequencingSteps of genome sequencing
DNA sequencing approachesDNA sequencing approaches
• The method preferred by the Human Genome Project is the hierarchical shotgun sequencing method.
• Also known as – The Clone-by-Clone Strategy– the map-based method– map first, sequence later
– top-down sequencing
Hierarchical Shotgun Sequencing Hierarchical Shotgun Sequencing
Human Genome Project adopted a map-based strategy– Start with well-defined physical map– Produce shortest tiling path for large-insert clones– Assemble the sequence for each clone
– Then assemble the entire sequence, based on the physical map
1) Markers for regions of the genomes are identified.
2) The genome is split into larger fragments (50-200kb) using restriction/cutting enzymes that contain a known marker.
3) These fragments are cloned in bacteria (E. coli) using BACs (Bacterial Artificial Chromosomes), where they are replicated and stored.
4) The BAC inserts are isolated and the whole genome is mapped by finding markers regularly spaced along each chromosome to determine the order of each cloned.
5) The fragments contained in these clones have different ends, and with enough coverage finding a scaffold of BAC contigs. This scaffold is called a tiling path. BAC contig that covers the entire genomic area of interest makes up the tiling path.
6) Each BAC fragment in the Golden Path is fragmented randomly into smaller pieces and these fragments are individually sequenced using automated Sanger sequencing and sequenced on both strands.
7) These sequences are aligned so that identical sequences are overlapping. Assembly of the genome is done on the basis of prior knowledge of the markers used to localize sequenced fragments to their genomic location. A computer stitches the sequences up using the markers as a reference guide.
In The Clone-by-Clone StrategyIn The Clone-by-Clone Strategy
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Fig: Hierarchical shotgun sequencing
In this approach, every part of the genome is actually sequenced roughly 4-5 times to ensure that no part of the genome is left out.
The Clone-by-Clone Strategy used in S. cerevisiae (yeast), C. elegans (nematode), Arabidopsis thaliana (mustard weed), Oryza sativa, Drosophila melanogaster and Homo sapiens (Human), etc.
Each 150,000 bp fragment is inserted into a BAC (bacterial artificial chromosome). A BAC can replicate inside a bacterial cell. A set of BACs containing an entire human genome is called a BAC library.
The Clone-by-Clone StrategyMarkers used in mapping large genomes
The Clone-by-Clone StrategyMarkers used in mapping large genomes
Different types of Markers are used in mapping large genomes, Such as
A. Restriction Fragment Length Polymorphisms (RFLP)
B. Variable Number of Tandem Repeats (VNTRs)
C. Sequence Tagged Sites (STS)
D. Microsatellites, etc.
A. Restriction Fragment Length Polymorphisms (RFLP)
Polymorphism means that a genetic locus has different forms, or alleles.
The cutting the DNA from any two individuals with a restriction enzyme may yield fragments of different lengths, called Restriction Fragment Length Polymorphisms (RFLP), is usually pronounced “rifflip”.
The pattern of RFLP generated will depend mainly on
– 1) The differentiation in DNA of selected strains (or) species– 2) The restriction enzymes used– 3) The DNA probe employed for southern hybridization
Steps: a. Consider the restriction enzyme HindIII, which recognizes the sequence
AAGCTT.
b. Between two, One individual contains three sites of a chromosome, so cutting the DNA with HindIII yields two fragments, 2 and 4 kb long.
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Figure: Detecting a RFLP
c. Another individual may lack the middle site but have the other two, so cutting the DNA with HindIII yields one fragment 6 kb long. These fragments are called RFLP.
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d. These restriction fragments of different lengths beteween the genotypes can be detected on southern blots and by the use of suitable probe. An RFLP is detected as a differential movement of a band on the gel lanes from different species and strains. Each such bond is regarded as single RFLP locus. So any differences among the DNA of individuals are easy to see.
e. This RFLP is used as a marker in chromosomal mapping.
Limitations Requires relatively large amount of highly pure DNA Laborious and expensive to identify a suitable marker restriction
enzymes. Time consuming. Required expertise in auto radiography because of using radio actively
labeled probes
B. Variable Number of Tandem Repeats (VNTRs)Due to the greater the degree of polymorphism of a RFLP, mapping
become very tedious, in this case variable number tandem repeats (VNTRs) will be more useful.
Tandem repeats occur in DNA when a pattern of one or more nucleotides is repeated and the repetitions are directly adjacent to each other.
An example would be:
In which the sequence ATTCGCCAATC is repeated three times.
• A variable number tandem repeat (or VNTR) is a location in a genome where a short nucleotide sequence is organized as a tandem repeat.
• The repeated sequence is longer — about 10-100 base pairs long.• The full genetic profiles of individuals reveal many differences. • Since most human genes are the same from person to person, but
Variable Number of Tandem Repeats or VNTRs that tends to differ among different people.
ATTCGCCAATC ATTCGCCAATC ATTCGCCAATCATTCGCCAATC ATTCGCCAATC ATTCGCCAATC
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• While the repeated sequences themselves are usually the same from person to person, the number of times they are repeated tends to vary.
• VNTRs are highly polymorphic. These can be isolated from an individual’s DNA and therefore relatively easy to map.
• However, VNTRs have a disadvantage as genetic markers: They tend to bunch together at the ends of chromosomes, leaving the interiors of the chromosomes relatively devoid of markers.
C. Sequence Tagged Sites (STS)
Another kind of genetic marker, which is very useful to genome mappers, is the sequence-tagged site (STS). •STSs are short sequences, about 60–1000 bp long, that can be easily detected by PCR using specific primers. •The sequences of small areas of this DNA may be known or unknown, so one can design primers that will hybridize to these regions and allow PCR to produce double stranded fragments of predictable lengths. If the proper size appears, then the DNA has the STS of interest.•One great advantage of STSs as a mapping tool is that no DNA must be cloned and examined. •Instead, the sequences of the primers used to generate an STS are published and then anyone in the world can order those same primers and find the same STS in an experiment that takes just a few hours.
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In this example, two PCR primers (red) spaced 250 bp apart have been used. Several cycles of PCR generate many double-stranded PCR products that are precisely 250 bp long. Electrophoresis of this product allows one to measure its size exactly and confirm that it is the correct one.
Figure : Sequence-tagged sites
1. Geneticists interested in physically mapping or sequencing a given region of a genome aim to assemble a set of clones called a contig, which contains contiguous (actually overlapping) DNAs spanning long distances.
2. It is essential to have vectors like BACs and YACs that hold big chunks of DNA. Assuming we have a BAC library of the human genome, we need some way to identify the clones that contain the region we want to map.
3. A more reliable method is to look for STSs in the BACs. It is best to screen the BAC library for at least two STSs, spaced hundreds of kilo-bases apart, so BACs spanning a long distance are selected.
4. After we have found a number of positive BACs, we begin mapping by screening them for several additional STSs, so we can line them up in an overlapping fashion as shown in following figure. This set of overlapping BACs is our new contig. We can now begin finer mapping, and even sequencing, of the contig.
Making physical map using Sequence Tagged Sites (STS)
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At top left, several representative BACs are shown, with different symbols representing different STSs placed at specific intervals. In step (a) of the mapping procedure, screen for two or more widely spaced STSs. In this case screen for STS1 and STS4. All those BACs with either STS1 or 4 are shown at top right. The identified STSs are shown in color. In step (b), each of these positive BACs is further screened for the presence of STS2, STS3, and STS5.The colored symbols on the BACs at bottom right denote the STSs detected in each BAC. In step (c), align the STSs in each BAC to form the contig. Measuring the lengths of the BACs by pulsed-field gel electrophoresis helps to pin down the spacing between pairs of BACs.
Fig: Mapping with STSs.
D. Microsatellites STSs are very useful in physical mapping or locating specific sequences in the genome. But sometimes it is not possible to use them for genetic mapping. Fortunately, geneticists have discovered a class of STSs called microsatellites.
GCTTGGTGTGATGTAGAAGGCGCCAATGCATCTCGACGTATGCGTATACGGGTTACCCCCTTTGCAATCAGTGCACACACAC
ACACACACACACACACACACACACACACACAGTGCCAAGCAAAAATAACGCCAAGCAGAACGAAGACGTTCTCGAGAACACC
GCTTGGTGTGATGTAGAAGGCGCCAATGCATCTCGACGTATGCGTATACGGGTTACCCCCTTTGCAATCAGTGCACACACAC
ACACACACACACACACACACACACACACACAGTGCCAAGCAAAAATAACGCCAAGCAGAACGAAGACGTTCTCGAGAACACC
Microsatellites are similar to minisatellites in that they consist of a core sequence repeated over and over many times in a row.
The core sequence in typical microsatellites is smaller—usually only 2–4 bp long.
Microsatellites are highly polymorphic; they are also widespread and relatively uniformly distributed in the human genome.
The number of repeats varied quite a bit from one individual to another. Thus, they are ideal as markers for both linkage and physical mapping.
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In 1992, Jean Weissenbach et al produced a linkage map of the entire human genome based on 814 microsatellites containing a C–A dinucleotide repeat.
The most common way to detect microsatellites is to design PCR primers that are unique to one locus in the genome and unique on base pair on either side of the repeated portion.
Therefore, a single pair of PCR primers will work for every individual in the species and produce different sized products for each of the different length microsatellites.
The PCR products are then separated by either gel electrophoresis. Either way, the investigator can determine the size of the PCR product and thus how many times the dinucleotide ("CA") was repeated for each allele.
Whole genome Shotgun SequencingWhole genome Shotgun SequencingThe shotgun-sequencing strategy, first proposed by Craig Venter,
Hamilton Smith, and Leroy Hood in 1996, bypasses the mapping stage and goes right to the sequencing stage.
This method was employed by Celera Genomics, which was a private entity that was trying to mono-polise the human genome sequence by patenting it, to do this they had to try and beat the publicly funded project. Whole genome shotgun sequencing was therefore adopted by them.
1. BAC library: A BAC library is generated of random fragments of the human genome using restriction digestion followed by cloning.
The sequencing starts with a set of BAC clones containing very large DNA inserts, averaging about 150 kb. The insert in each BAC is sequenced on both ends using an automated sequencer that can usually read about 500 bases at a time, so 500 bases at each end of the clone will be determined.
Assuming that 300,000 clones of human DNA are sequenced this way, that would generate 300 million bases of sequence, or about 10% of the total human genome. These 500-base sequences serve as an identity tag, called a sequence-tagged connector (STC), for each BAC clone. This is the origin of the term connector—each clone should be “connected” via its STCs to about 30 other clones. Continue………
Fig: Whole Genome Shotgun Sequencing Method
Steps:1. BAC library
2. Finger printing
3. Plasmid library
4. BAC walking
5. Powerful computer program
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2. Finger printing: This step is to fingerprint each clone by digesting it with a restriction enzyme. This serves two important purposes. First, it tells the insert size (the sum of the sizes of all the fragmented by the restriction enzyme). Second, it allows one to eliminate aberrant clones whose fragmentation patterns do not fit the consensus of the overlapping clones. Note that this clone fingerprinting is not the same as mapping; it is just a simple check before sequencing begins. 3. Plasmid library: A seed BAC is selected for sequencing. The seed BAC is sub cloned into a plasmid vector by subdividing the BAC into smaller clones only about 2 kb. A plasmid library is prepared by transforming E. coli strains with plasmid. This whole BAC sequence allows the identification of the 30 or so other BACs that overlap with the seed: They are the ones with STCs that occur somewhere in the seed BAC.
4. BAC walking: Three thousand of the plasmid clones are sequenced, and the sequences are ordered by their overlaps, producing the sequence of the whole 150-kb BAC. Finding the BACs (about 30) with overlapping STCs, then compare them by fingerprinting to find those with minimal overlaps, and sequence them. This strategy, called BAC walking, would in principle allow one laboratory to sequence the whole human genome. Continue………
5. Powerful computer program: But we do not have that much time, so Venter and colleagues modified the procedure by sequencing BACs at random until they had about 35 billion bp of sequence. In principle that should cover the human genome ten times over, giving a high degree of coverage and accuracy. Then they fed all the sequence into a computer with a powerful program that found areas of overlap between clones and fit their sequences together, building the sequence of the whole genome.
Finishing
• Process of assembling raw sequence reads into accurate contiguous sequence– Required to achieve
1/10,000 accuracy
• Manual process– Look at sequence reads at
positions where programs can’t tell which base is the correct one
– Fill gaps– Ensure adequate coverage
GapSingle
stranded
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Finishing
• To fill gaps in sequence, design primers and sequence from primer
• To ensure adequate coverage, find regions where there is not sufficient coverage and use specific primers for those areas
GAP
Primer
Primer
Verification
• Region verified for the following:– Coverage– Sequence quality– Contiguity
• Determine restriction-enzyme cleavage sites – Generate restriction map of sequenced region– Must agree with fingerprint generated of clone
during mapping step
New technologiesNew technologies
• A high-priority goal at the beginning of the Human Genome Project was to develop new mapping and sequencing technologies
• To date, no major breakthrough technology has been developed– Possible exception: whole-genome shotgun sequencing applied
to large genomes, Celera
Automated sequencersAutomated sequencers• Perhaps the most important contribution to large-
scale sequencing was the development of automated sequencers– Most use Sanger sequencing method– Fluorescently labeled reaction products– Capillary electrophoresis for separation
Automated sequencers: ABI 3700
96–well plate
robotic arm and syringe
96 glass capillaries
load bar
MegaBACE ABI 3700
Automatic gel reading
Computer image of sequence read by
automated sequencer
Sequence assembly readout
Consensus building
Genome sequencing achievment in Bangladesh
Genome sequencing achievment in Bangladesh
• Genome sequencing of Macrophomina phaseolina • Genome sequencing of Jute
Genome of destructive Pathogen Macrophomina phaseolina unraveled by Maqsudul Alam & BJRI Associates
Genome of destructive Pathogen Macrophomina phaseolina unraveled by Maqsudul Alam & BJRI Associates
Macrophomina phaseolina is a soil and seed borne fungus.
it can infect more than 500 cultivated and wild plant species.
It causes seedling blight, dry root rot, wilt, leaf blight, stem blight, root and stem rot of different cultivated and wild plant species.
The fungus can remain viable for more than 4 years in soil and crop.
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• The Basic and Applied Research on Jute (BARJ) project team, led by Prof Maqsudul Alam, took this unique challenge and, for the first time in the world, decoded the genome of this most dangerous fungus.
• They have identified the proteins and their networks that the fungus uses to attack and kill the plant. This fundamental knowledge will help to defend and fight against this fungus and to promote the development of resistant varieties of jute as well as other crops.
• Jute was called the Golden Fiber of Bangladesh as Bangladesh was the largest jute production country of the world.
• Genome sequencing of jute has been discovered by Bangladeshi scientists.
Genome sequencing of Tossa jute (Corchorus olitorius)Genome sequencing of Tossa jute (Corchorus olitorius)
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• The country first time in world decoded the jute genome.• The research team was led by Professor Maqsudul Alam from University of
Hawaii, who also successfully led the genome discovery of papaya in USA and rubber in Malaysia.
This was done under the Basic & Applied Research on Jute Project (BARJ).
• Also included a group of Bangladeshi researchers from Dhaka
University's Biochemistry and Biotechnology departments,
Bangladesh Jute Research Institute (BJRI) software firm Data Soft in collaboration with Centre
for Chemical Biology, University of Science, Malaysia and University of Hawaii have successfully decoded
the jute's genome.
Fig: Internationally famed geneticist Maqsudul Alam and
other scientists of jute genome project
Anticipated Benefits of Genome ResearchAnticipated Benefits of Genome Research
Molecular Medicine • improve diagnosis of disease• detect genetic predispositions to disease• create drugs based on molecular information
Microbial Genomics• rapidly detect and treat pathogens (disease-causing microbes) in clinical practice• develop new energy sources (biofuels)• monitor environments to detect pollutants• clean up toxic waste safely and efficiently.
Risk Assessment • evaluate the health risks faced by individuals who may be exposed to radiation and to cancer-causing chemicals and toxins
Bio-archaeology, Anthropology, Evolution
• study evolution through mutations in lineages• study migration of different population groups based on maternal inheritance Continue………
• compare breakpoints in the evolution of mutations with ages of populations and historical events.
Agriculture, Livestock Breeding, and Bio-processing
• grow disease-, insect-, and drought-resistant crops• breed healthier, more productive, disease-resistant farm animals• grow more nutritious produce• develop biopesticides• incorporate edible vaccines incorporated into food products
DNA Identification (Forensics)
• identify potential suspects whose DNA may match evidence left at crime scenes• identify crime victims• establish paternity and other family relationships• identify endangered and protected species as an aid to wildlife officials • detect bacteria and other organisms that may pollute air, water, soil, and food• match organ donors with recipients in transplant programs
References • Weaver RF 2005. Molecular Biology. McGraw-Hill
International edition, NY.• Gardner EJ, MJ Simmons and DP Snustad 1991.
Principles of Genetics. John Wiley and Sons Inc, NY.
• Gupta, P.K. 2007. Genetics. Rastogi Publications, Meerut.
• Allison LA, 2007. Fundamental Molecular Biology, Blackwell publishing, USA
• Internet
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