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8/3/2019 Microbial Genome Sequencing Projects
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Presented by,
Devarapalli Pratap,Department of Genomic Science
Central University of Kerala
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MICROBIAL GENOME
It is the entirety of an microorganism's hereditary information
which is encoded either inDNAor, for many types of viruses, in
RNA. The genome includes both the genes and thenon-codingsequences of DNA/RNA.
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History of microbial genomesequencing projects
1977 - first complete genome sequenced was
bacteriophage X174 5,386 bp
First genome sequenced using random DNA
fragments Bacteriophage - 48,502 bp
1986 - mitochondrial (187 kb) and chloroplast (121 kb)genomes ofMarchantia polymorphaaresequenced
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Early 90s - cytomegalovirus(229 kb) and
Vaccinia(192 kb)genomes are sequenced
1995 - first complete genome sequence from a free living
organism - Haemophilus influenzae(1.83 Mb) Late 1990s - many additional microbial genomes were
sequenced includingArchaea (Methanococcus
jannaschii - 1996)and Eukaryotes (Saccharomyces
cerevisiae - 1996)
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Microbial genomes sequenced to date
As per the current update 7795 microbes are selected for
sequencing in which1710 microbial genomes are
completely sequenced, 2325 are in the stage ofassemblyand3760 areunfinished.
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Microbial GenomeSequencing Project
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STEPS INVOLVED
Library construction
Genome sequencing
Assembly phaseAnnotation
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Both conventional and large insert genomic DNA libraries
should be constructed.
The small insert library will be used for the bulk of thesequencing in order to generate suitable coverage of the
complete genome.
The large insert library (YAC, BAC, PAC, cosmid etc.) will beused during the sequence closure phase.
Library Construction
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Ordered Clone approach
This uses a large insert library to construct a map of
overlapping clones covering the whole genome. Selected clones
are then sequenced to obtain the whole genome sequence.
Random sequencing approach
Direct short gun sequencing which does not require preliminary
data .
Genome sequencingstrategies
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Ordered Clone approach
Whole Genome
Large
DNAfragment Digest and
subclone
Randomly
sequence
fragments
Fill gaps
Repeat for entire genome map
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Random sequencing approach
Whole Genome
Shear and
subclone
Randomly
sequence
fragments
Fill gaps
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Assembly Phase
The assembly phase is composed of three major steps :
The conversion of the data from automated sequencers tonucleotide sequences.
The utilization of these sequences in the assembly process.
The continuous assessment of this assembly process.
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AutomatedSequencers
Phred
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PhredPhred is a base-calling program for DNA sequence traces. Phredreads DNA sequence chromatogram files and analyzes the peaks tocall bases, assigning ("Phred scores") to each base call.
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Sequence assembly and Gap closure
Random sequences initially interpreted using highly accurate basecalling software and assembled to generate primary contigs** usingsoftware such as Phrap.
Computational and experimental techniques used to identify linkingclones and order primary contigs**
Primer walk sequencing of linking clones and PCR products to fillsequence gaps between contigs**
Confirmation of contig** order by PCR
**The DNA sequence reconstructed from a set of overlapping DNA segments
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Linking Clones
Those gaps with suitable linking clones can be confirmed by
PCR and closed by primer walk sequencing
**Primer walking is a sequencing method of choice for sequencing DNA
fragments between 1.3 and 7 kilo bases.
Contig 1Contig 2
FWD REV
Large Insert Linking Clone
Primer Walking**
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Physical Gaps
Peptides can link the coting ends having regions withhomology to the same gene (or operon / gene cluster)
Physical gaps can also be filled with the help of Southern
Hybridization.
Contig 2 Contig 6
FWD REV
PCR Product
Linked by Southern Hybridization
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Annotation
SequenceGene prediction
ProteinsSimilarity searches againstreference databases Calculations & predictions(MW , structure, location etc)
Annotated Proteins
Pathway predictionAnnotated Proteins &pathways
Data GenerationManual editing
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Completely Sequenced Microbial
Genome
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Over View
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Conclusion
Microbial genome sequencingand analysis is a rapidly
expanding and increasingly important strand of microbiology,
Important information about the specific adaptations and
evolutionof a microorganism can be determined from genome
sequencing. However, genome sequencing merely a strong
starting point on road to completely understand the biology ofmicroorganisms
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References
Lionel .F., Etal .,(1999) .Cloning and assembly strategies inmicrobial genome projects. Microbiology Res145, 26252634.
Claire M. F., Jonathan A. E. and Steven L. S.,(2000).Microbialgenome sequencing.Nature Res 406, 799-803.
Karen E. N., Ian T. P., Heidelberg J. F., and Claire M. F.,(2000).Status of genome projects for nonpathogenic bacteria and
archaea. Nature Biotechnology Res18, 1049-1054. Uchiyama. I.,(2003).microbial genome database for
comparative analysis. Nucleic Acids Research Res31,58-62.
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Presented by,D evarapalli P ratap