Microbial Genome Sequencing Projects

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Text of 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