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sophie nicaud — 2006-11-28T19:45:57Z

Sequencing strategies
At present, there are two strategies for sequencing large genomes :

Whole genome shotgun sequencing
Clone-by-clone sequencing

Sequencing steps
In the context of large whole genome projects, the production of sequence data at Genome is performed as a four-step process :

Extraction of DNA
Cloning
Sequencing
Annotation of the sequences

FAQ

sophie nicaud — 2006-11-28T19:46:47Z

sophie nicaud — 2006-11-28T19:49:55Z

Parliamentary office for the evaluation of scientific and technological choices (link)
Public hearing of March 28, 2007

In the context of the contributions of science and technology to sustainable development, Senators Pierre Lafitte and Claude Saunier invited several scientific personalities to respond to their questions. Jean Weissenbach, Director of Genoscope, participated in the Round Table, “Sustainable development of biodiversity: How biodiversity can can continue to be a source of the indispensable and diversified services furnished by ecosystems, and become the future toolbox for the 4th industrial revolution?” The following is an extract of his intervention:
“...I will mainly discuss the importance of microbial diversity in a certain number of applications, notably in the field of chemistry. As has been emphasized, we still do not know the number of microbial species which exist. Nevertheless, this figure is not as important as the biological functions that these microbes are capable of performing. Here again we have no inventory; this will require a considerable scientific effort. A few days ago Craig Venter published a list of seven million genes of microbial origin; we do not know the function of half of these, although it is likely that they are involved in bioconversions. These bioconversions are essential for the chemistry of the future. Since these bioconversions will not longer be based on petroleum, this will necessitate the the use of catalysts for a complete series of chemical reactions that at present can only be carried out by nature. When we ask chemists to use enzymes, they very often reply that they know nothing about them. In fact, when I heard this type of reply a few weeks ago during a meeting of the National Research Agency, I was particulary disturbed to note that our chemist colleagues are still thinking in very classical terms. It is the same with our industrialists—the only way to to convince them was to propose “turnkey” solutions, i.e. to place all the research efforts on the scientists. However, at present the situation of biochemical research in France is cause for concern. Biochemistry has effectively been replaced by molecular biology, which I represent. At the moment, students don't want to hear about biochemistry or metabolism. It is therefore essential for us to reverse this tendancy, by taking inspiration from our neighbors like Germany, which still has a very satisfactory level of biochemistry. Also, its chemical industry is paying a lot of attention to bioconversion. It is the same in the United States. We must therefore make major efforts to encourage the industrialists and the political institutions to relaunch research in these domains.”

Actes de l'audition du 28 mars 2007

Photo gallery

sophie nicaud, ssamair — 2007-09-25T09:22:19Z

Genoscope - Cutting of a gel band containing fragments of DNA to be sequenced, of defined size. These fragments will be inserted into a “vector” which will be used to transform the bacterium which will be used to amplify them

Glossary

sophie nicaud — 2007-09-25T09:24:28Z

Alleles
Different versions of the sequence of the same gene.

Amino acid
See Protein

Annotation
Once the DNA has been sequenced, annotation consists of defining the parts of the sequence which correspond to genes (instructions) in the sequence in silico, and of obtaining an idea about their function. Various resources are utilized, including computerized prediction programmes which are not infallible (this is called ab initio annotion when theses programmes are used alone), comparisons with other genomic sequences, with cDNA sequences or with proteins from the same organism or different organisms. (See the sections on Interpretation of sequences, Comparing genomes, Complementary DNA, Annotation of the human genome).

Base
The variable unit of nucleotides. There are 4 types of bases in DNA: thymine (T), cytosine (C), adenine (A) and Guanine (G). In RNA, thymine is replaced by uracil (U). The order of the bases along the DNA molecule constitutes the sequence. The bases which are opposite each other on each strand of DNA are paired according to the rules, A—T and G---C. These rules make it possible to define the complementary sequence of a specific sequence.

Chromosome
Genomes are organized as DNA molecules called chromosomes. Bacterial cells contain a single chromosome, whereas the genomes of higher organisms (plants, animals) include various numbers of chromosomes, the same number for each species; each chromosome is generally present in two copies which are not identical; one copy is inherited from the mother and the other from the father. In humans there are 23 pairs of chromosomes including one pair of sex chromosomes which are different in men (X and Y) and the same in women (XX). There are thus 24 types of human chromosomes, which are numbered more or less according to size. During cell division, DNA molecules which are associated with proteins, condense and the chromosomes appear as rods visible in the microscope.

Cloning
In molecular biology this word designates identical reproduction of a DNA fragment which is integrated into a bacterium and recopied by it as if it were its own DNA. After spreading over a Petri dish, each bacterial clone—the totality of cells originating from the division of a single bacterial cell—form colonies. This is the means used for isolating DNA fragments from a mixture, to conserve them and prepare them in large quantity. The word clone is also used to designate the fragment of DNA present which is reproduced infinitely in a bacterial clone.

Complementary DNA (cDNA)
Molecule of DNA obtained by copying a molecule of messenger RNA (mRNA). In contrast to RNA, the molecule of cDNA can be cloned and amplified, and thus provides access to the sequence of the mRNA. Its name comes from the fact that one of the strands of DNA is complementary to the RNA strand.

DNA
Deoxyribonucleic acid (DNA) is the molecule which contains the genetic information. This information is encoded by a chain, or sequence, of nucleotides, which are the basic subunits of DNA. A molecule of DNA is formed of two chains with complementary sequences, paired and twisted into a double helix. This structure was elucidated by James Watson and Francis Crick in 1953.

Electrophoresis
A procedure which is used to separate molecules (DNA in the case of sequencing) according to their size. The separation takes place on a gel—which traps water in a polymer, and permits molecules to migrate in an electric field. Small molecules migrate faster than the large ones, which are slowed down. Molecules of DNA which differ in length by a single nucleotide can be separated on these gels.

Exon
The majority of genes in plants and animals are fragmented. The biologically significant parts, the exons, are separated by intervening sequences without significance, the introns, which are eliminated in the messenger RNA. Finding the exon-intron boundaries is one of the major problems of annotation.

Gel
See Electrophoresis.

Gene
The functional unit of the genome. A gene corresponds to an instruction to be followed by the cell. The genome of a bacterium contains several thousand genes; that of plants or vertebrates (and therefore humans) consists of about 30 000 genes. Some genes code for RNA molecules with crucial functions in the cell, but the majority encode the sequences of proteins, which are synthesized via messenger RNA molecules (See RNA).

Genome
The ensemble of the genetic information of an organism present in each of its cells. The material substance of genome consists of DNA (except in some viruses, which have RNA as their genetic material) in the form of long molecules called chromosomes.

Insert
See Vector

In silico
Used to describe an operation which is performed using a computer..

Intron
See Exon.

Nucleotide
Nucleotides are the molecules (links) which form the strands of the DNA chain. A nucleotide consists of a constant part—a sugar and a phosphate—and a variable part, a nitrogenous base, which exists in 4 forms (see Base).

PCR
Polymerase chain reaction, is a reaction catalyzed by the enzyme Taq polymerase, which amplifies DNA fragments for sequencing or other purposes.

Read
This term designates a single sequencing operation as well as the information it delivers, i.e. a sequence which is 800-1000 bases long, generally from one of the extremities of a longer DNA fragment.

Restriction enzymes
Enzymes which are capable of cutting DNA molecule at the sites of specific sequences. There are more than 400 of these, which recognize sequences from 4 to 8 bases in length. The longer sequences are the least frequent in the genome, and the enzymes which recognize these sites can cut the DNA into large fragments. Restriction enzymes are also used to establish specific “profiles” of large fragments (fingerprints). Comparison of these profiles is used to identify overlapping fragments.

RNA
Ribonucleic acid is a molecule which is very similar to DNA, but consists of a single chain of nucleotides. The instructions encoded in the DNA sequence are copied (“transcribed”) into a type of RNA called messenger RNA, for translation into proteins, which are the real agents of cellular functions. These RNA molecules are also called transcripts.

Sequence, sequencing
Sequencing is the operation which consists of determining the order, or sequence, of the bases along the DNA molecule. A sequence looks like a text of variable length, written with an alphabet of only 4 letters, A, T, C and G (see Base). Genoscope determined the order of the 87 million letters of the uninterrupted sequence of human chromosome 14.

Template
The templates are DNA molecules which contain an insert to be sequenced, which has been extracted and purified from a bacterial clone. They are then ready to be used in sequencing reactions or PCR.

Transformation
An operation which consists of using a vector to insert a DNA sequence which it contains into a bacterium in order to clone this sequence (insert). Very brief electric shocks are used for this purpose today, with good efficiency.

Vector
Small circular molecules of DNA present in bacteria next to the bacterial chromosome. When removed from bacteria they are utilized for the molecular cloning of the DNA fragment to be studied. After the fragment has been inserted (then called an insert), the vector is re-introduced into a bacterial cell for transformation.

General informations

sophie nicaud — 2007-09-25T09:25:32Z

	Thesis & Seminars
	About sequencing
	Answers to the questions most frequently put on the Genome and the DNA, the sequencing and on the project human Genome
	Photo gallery on the activity of sequencing in Genoscope
	The indispensable definitions in molecular biology and in genomics

Sequencing strategies

sophie nicaud — 2008-01-08T13:45:10Z

Introduction
Whole genome shotgun sequencing
Clone-by-clone sequencing

Introduction

The sequencing of large genomes remains an arduous enterprise, for which several strategies exist. All these strategies encounter the same problem: since sequencing machines can only deliver 1000 nucleotides per read at best, how can the ensemble of the genome sequence, which is thousands (bacteria) to millions (mammals) of times longer, be reconstituted?

Introduction
Whole genome shotgun sequencing
Clone-by-clone sequencing

Whole genome shotgun sequencing

The “rough” assembly of sequences from a genome constitutes a strategy called whole genome shotgun sequencing. This method works well for bacterial genomes, which are small (of the order of several million nucleotides) and do not have many repetitive sequences. The contribution of this strategy to the sequencing of the human genome, which consists of 3 billion nucleotides, half of which are repetitive, remains controversial (see the Human Genome section).

In the case of large genomes, the whole genome shotgun strategy involves obtaining matching sequences at different genomic levels. Thus the ends of large genomic fragments (several hundred thousands of nucleotides) are sequenced, which allows assembly of contigs as large backbones, and permits detection of certain errors within the contigs.

Introduction
Whole genome shotgun sequencing
Clone-by-clone sequencing

Clone-by-clone sequencing

A different strategy, called “clone-by-clone” or hierarchic sequencing, has been adopted by several international consortia for the sequencing of the human genome and other large genomes (rice, Arabidopsis, roundworm, etc.). The assembly of overlapping reads is no longer performed at the scale of the ensemble of the genome, but at the scale of large genomic fragments (wrongly called “clones”), which are first ordered on a map. This compartmentalization reduces the difficulties due to repetitive sequences, and also makes it possible to target the “finishing” work to precise regions. This finishing step is indispensable in the perspective of an exhaustive and precise annotation of the sequence.

On the other hand, a major mapping effort is needed to put the large genomic fragments (see the Mapping section). This work requires diverse resources: markers from physical and genetic maps, assembly of large overlapping fragments based on their restriction enzyme profile (“fingerprint”), and finally, the end sequences of the large fragments. This last resource may be used as a step-by-step method to construct the map of large fragments; as the sequencing progresses, one can choose overlapping fragments which are least redundant for sequencing.

With a volume of sequences to be read equal to 5 or 6 times the size of a genome, such as that of humans (5X coverage), it is possible to assemble a “draft” clone-by-clone sequence in which several dozen contigs are obtained for each clone. These initial contigs are generally not ordered or oriented. By increasing the coverage to 10X, longer and fewer contigs are obtained, and ordered and oriented thanks to paired reads or other information. This must be followed by fastidious polishing and finishing steps, to ensure less than one error per 10,000 nucleotides, and to fill the residual gaps by working in the specific local regions.

Sequencing steps

sophie nicaud — 2008-01-11T09:07:02Z

(Click the image to view the document)

List of FAQs

sophie nicaud — 2008-01-11T10:16:00Z

1: What is DNA?
2: What is a genome?

1: What is the public project for sequencing the human genome?
2: Has the human genome been completely sequenced?
3: How many genes do humans have?
4: Why is it so difficult to find the genes in a human genome sequence?
5: Where did the sequenced human DNA come from?
6: Is the human genome “freely available”? If not, who owns it?
7: Why was there a Human Genome Project? What is its use?
8: Who were the members of the international consortium? What was the role of each of them?

1: What is the DNA sequence?
2: Why do we sequence DNA?
3: How do we sequence DNA?
4: What is the assembly?
5: Why were Genome Centers created?

Genome & DNA

sophie nicaud — 2008-01-11T11:17:56Z

1: What is DNA?
2: What is a genome?

Deoxyribonucleic acid (often abbreviated as DNA) is a molecule which is found in all living organisms. The DNA is present in the nucleus of eukaryotic cells, in the cytoplasm of prokaryotic cells, and in the matrix of both mitochondria and chloroplasts. Some viruses also have DNA encapsulated in their capsids. DNA is considered to be the molecule of heredity because it constitutes the genome of living organisms and is transmitted in totality or in part during the process of reproduction. It is the basis for the synthesis of proteins.

1: What is DNA?
2: What is a genome?

The word genome designates the ensemble of hereditary information of an organism. This information is present in totality in every cell of the organism (1). When a cell divides, this information is transmitted to the daughter cells.

The genome contains all the instructions necessary for the development, function, reproduction and maintenance of the integrity of cells and organisms. These instructions are called genes.

The material support for the genetic information is DNA (deoxyribonucleic acid). The genome is composed of giant DNA molecules associated with other types of molecules named proteins to form the chromosomes. A human being possesses 23 pairs of chromosomes, i.e. two complete sets of instructions, each of which is inherited from one of her/his parents (2).

Two living organisms of different species present genomes which differ in their size and in their number, and in the order and the nature of the instructions which they contain. Two individuals of the same species, on the other hand, possess the same catalog of instructions, even though they may exist in versions which are slightly different (from one individual to the next, or in a single individual when the copies inherited from the father and the mother are different). It is in this sense that we speak of the human genome, shared by all human beings with its unique gene baggage. In the strict sense, the genome of each human being is unique (with the exception of those of identical twins), but it only differs by 0.1% from that of an unrelated individual.

The size of genomes, measured in number of bases (see “What is a DNA sequence?”), is extremely variable: several tens of thousands of bases on average for a virus genome, several million bases for a bacterium, three billion bases for the human genome—and 16 billion for the wheat genome! The number of genes contained in the genomes varies less: several thousand in a bacterium, 13 000 in Drosophila, 25 000 in humans. It is difficult to correlate the complexity of organisms with their number of genes, and even more difficult to correlate the size of their genomes. There are lots of exceptions.

(1)With a few exceptions, such as human red blood cells
(2)Individuals of the masculine sex possess one pair of dissimilar sex chromosomes, which have a different set of instructions. Furthermore abnormalities in the number of chromosomes exist, such as trisomy 21.

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Sequencing steps

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Genome & DNA