10. 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 technologies
Genome sequencing achievment in Bangladesh
Benefits of Genome Research
At a glance
11. 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.
Continue………
12. 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.
13. How many types of genomes are:
1. Prokaryotic Genomes
2. 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?
14. Origin 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
• the structure and function of entire genomes
• mapping,
• sequencing ,
• and analyzing the functions of entire genomes
The field includes studies of intro-genomic phenomena
such as heterosis, epistasis, pleiotropy and other interactions
between loci and alleles within the genome.
15. 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.
16. Types of Genomics
1. 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.
17. 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 Sequencing
Challenges 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.
18. Milestones in Genomic Sequencing
1977; Fred Sanger; fX 174 bacteriophage (first sequenced genome );
5,375 bp
Amino acid sequence of phage proteins
Overlapping genes only in viruses
Fig: The genetic map of phage fX174 (Overlapping reading frames)
Continue………
19. 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
20. 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.
LogMW
Distance
. .
.
.
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.
21. 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 sequencing
DNA sequencing approaches
22. • 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
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
23. 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 Strategy
Continue………
24. 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.
25. 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.
26. The Clone-by-Clone Strategy
Markers 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.
27. 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.
Continue………
28. 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.
Continue………
29. 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
30. 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 ATTCGCCAATC
Continue………
31. • 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.
32. 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.
Continue………
33. 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
34. 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)
Continue………
35. 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.
36. 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.
GCTTGGTGTGATGTAGAAGGCGCCAATGCATCTCGACGTAT
GCGTATACGGGTTACCCCCTTTGCAATCAGTGCACACACAC
ACACACACACACACACACACACACACACACAGTGCCAAGCA
AAAATAACGCCAAGCAGAACGAAGACGTTCTCGAGAACACC
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.
Continue………
37. 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.
38. Whole genome Shotgun Sequencing
The 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………
40. 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………
41. 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.
42. 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
Gap
Single
stranded
Continue………
43. 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
44. 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
45. HISTORY OF DNA SEQUENCING
• 1972 – Earliest nucleotide sequencing – RNA sequencing of Bacteriophage
MS2 by WALTER FIESERR
• Early sequencing was performed with tRNA through a technique developed
by Richard Holley, who published the first structure of a tRNA in 1964.
• 1977 - DNA sequencing FREDRICK SANGER by Chain termination method
• Chemical degradation method by ALLAN MAXAM and WALTER GILBERT
• 1977 - First DNA genome t be sequenced of Bacteriophage ΦX174
• 1986 - LOREY and SMITH gave Semiautomated sequencing
• 1987 – Applied biosystems marketed Fully automated sequencing machines
46. Determining the Sequence of DNA
•Methods:
1) Maxam and Gilbert chemical degradation method
2) Chain termination or Dideoxy method
• Fredrick Sanger
3) Genome sequencing method
• Shotgun sequencing
• Clone contig approach
4) 2nd generation sequencing methods
• Pyrosequencing
• Nanopore sequencing
• Illumina sequencing
• Solid sequencing
47. SANGER SEQEUNCING
• Chain termination method of DNA sequencing.
• It involves following components:
a) 1. Primer
b) 2. DNA template
DNA polymerase
d) 4.. dNTPs(A,T,G,C)
e) 5. ddNTPs
• 4 Steps:
1. Denaturation
2. Primer attachment and extension of bases
3. Termination
4. Poly acrylamide gel electrophoresis
49. ddATP + ddA
four dNTPs dAdGdCdTdGdCdCdCdG
ddCTP + dAdGddC
four dNTPs dAdGdCdTdGddC
dAdGdCdTdGdCddC
dAdGdCdTdGdCdCddC
ddGTP + dAddG
four dNTPs dAdGdCdTddG
dAdGdCdTdGdCdCdCddG
ddTTP + dAdGdCddT
four dNTPs dAdGdCdTdGdCdCdCdG
A
C
G
T
Chain Termination (Sanger) Sequencing
51. SANGER’S METHOD
• Not all polymerases can be used as they have
mixed activity of polymerizing and degrading.
• Both exonuclease activities are detrimental.
• Klenow fragment was used in orignal method but it
has low processivity.
• So Sequenase from bacteriophage T7 was uesd
with high processivity and no exonuclease added.
• Method requires ss DNA. So it is obtained by
• Denaturation with alkali or boiling
• DNA can be cloned in phagemid containg M13 ori and
can take up DNA fragments of 10kb
52. PYROSEQUENCING
• Pyrosequencing is the second important type of DNA sequencing methodology
in use today.
• The addition of a DNTP is accompanied by release of a molecule of pyrophosphate.
• Reaction mixture contains
DNA sample to be sequenced
Primers
Deoxynucleotides
DNA polymerase
Sulfurylase
• The release of pyrophosphate is converted by the enzyme sulfurylase into a flash
of chemiluminescence which is easily automated.
53. Animated videos of Sanger’s and Pyrosequencing
methods for better understanding:
55. MASSIVELY PARALLEL PYROSEQUENCING
• The DNA is broken down into fragments between 300 to 500bp
• Each fragment is ligated with a pair of adaptor
• To attach to the beads
• Provide annealing sites for the primers for performing PCR
• Adaptors are attached to beads by biotin-streptavidin linkage
• Just one fragment becomes attached to one bead
• Each DNA fragment is now amplified using
• PCR is carried out in a oil emulsion, each bead residing within
own droplet in the emulsion
• Each droplet contains all the reagents for PCR and is physically
seprated from all the other droplets by the barrier provided by
the oil components in the emulsion.
• After PCR, the droplets are transferred on wells on plastic strip
and pyrosequencing reactions are carried out
56.
57. SHOTGUN SEQUENCING
• Shotgun sequencing, also known as shotgun cloning, is a method used
for sequencing long DNA strands or the whole genome.
•In shotgun sequencing, DNA is broken up randomly into numerous small segments
and overlapping regions are identified between all the individual sequences that are
generated.
• Multiple overlapping reads for the target DNA are obtained by performing several
rounds of this fragmentation and sequencing.
•Computer programs then use the overlapping ends of different reads to assemble
them into a continuous sequence.
•The shotgun approach was first used successfully with the bacterium Haemophilus
influenzae.
•Craig venter used this method to map the Human genome project in 2001.
59. Shotgun sequencing AND Next Generation Sequencing
ANIMATED VIDEOS for better understanding:
60. NEXT GENERATION SEQUENCING
• The concept behind NGS – the bases of small fragments
of DNAare sequentially identifed as signals emitted as
eachfragment is resynthesized from a dna template
strand
• NGS extends this process across millions of reactions in
a massively parallel fashion rather than being limited to
a single or a few dna fragments
74. SOLiD SEQUENCING
• The SOLiD instrument utilizes a series of ligation and detection rounds
to sequence millions of fragments simultaneously.
• There are five primer cycles performed on the instrument with each
cycle staggered by a single base and including a series of seven or ten
ligations for either a 35 or 50 base pair sequencing run.
• Each ligation decodes two bases and is recorded through fluorescent
imaging.
• By compiling the fluorescent reads in color space for each fragment, an
accurate sequence can be generated.
• Two types of libraries are available for sequencing Fragment and Mate
Pairs.
77. Conclusion:
The project was not able to sequence all the DNA found in human
cells. It sequenced only "euchromatic" regions of the genome,
which make up more than 95% of the genome. The other regions,
called "heterochromatic" are found in centromeres and telomeres,
and were not sequenced under the project.
The Human Genome Project was declared complete in April 2003.
An initial rough draft of the human genome was available in June
2000 and by February 2001 a working draft had been completed
and published followed by the final sequencing mapping of the
human genome on April 14, 2003. Although this was reported to
cover 99% of the euchromatic human genome with 99.99%
accuracy, a major quality assessment of the human genome
sequence was published on May 27, 2004 indicating over 92% of
sampling exceeded 99.99% accuracy which was within the intended
goal. Further analyses and papers on the HGP continue to occur.
Notas do Editor
Although automated editing programs like PHRAP have greatly increased the efficiency of sequencing, there remains a need for human judgment and intervention. This occurs during the finishing step, which is defined as the process of assembling the raw sequence reads into an accurate contiguous genomic sequence. For genomic sequencing with an accuracy of one error in 10,000 bases, a manual finishing step is essential. The finisher looks at positions where the automated editing program can’t tell which base is the correct one. By examining the various raw sequence reads, the finisher then makes a judgment call as to the correct base or sends the region back for additional sequencing. Similarly, when there are gaps in the sequence or insufficient coverage, the finisher will flag the region and send it back to the production sequencing team for more work.
Gaps are usually filled by designing custom sequencing primers that are complementary to the regions adjacent to the gap. The sequencing reaction is then performed using these custom primers on a clone containing the problematic region of DNA as the template. A similar strategy is used for regions with insufficient coverage: Custom primers are made and then used for directed sequencing of a particular region.
The final step of finishing is to verify the sequence. All regions are checked for the extent of coverage (i.e., how many times the same region has been sequenced, and in what direction), for sequence quality (i.e., whether ambiguity has been removed for all positions in the sequence), and for contiguity (i.e., whether the sequence forms one uninterrupted stretch of DNA). A good test of sequence quality that is frequently used in the finishing stage is to determine the sites where restriction enzymes would cut in the newly acquired sequence. A restriction map is generated from the sequence and then compared with the known fingerprint generated from the clone during the mapping step. If both show the same pattern, then it is considered to be an indication that the sequence is of high quality and relatively error free.