Presented by,

Devarapalli Pratap,
Department of Genomic Science Central University of Kerala

MICROBIAL GENOME
It is the entirety of an microorganism's hereditary information which is encoded either in DNA or, for many types of viruses, in RNA. The genome includes both the genes and the non-coding sequences of DNA/RNA.

History of microbial genome sequencing 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 of Marchantia polymorpha are sequenced

 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 including Archaea (Methanococcus jannaschii - 1996) and Eukaryotes (Saccharomyces cerevisiae - 1996)

Microbial genomes sequenced to date

As per the current update 7795 microbes are selected for sequencing in which 1710 microbial genomes are completely sequenced, 2325 are in the stage of assembly and 3760 are unfinished.

Microbial Genome Sequencing Project

STEPS INVOLVED
Library construction Genome sequencing Assembly phase Annotation

Library Construction
Both conventional and large insert genomic DNA libraries

should be constructed. The small insert library will be used for the bulk of the sequencing in order to generate suitable coverage of the complete genome. The large insert library (YAC, BAC, PAC, cosmid etc.) will be used during the sequence closure phase.

Genome sequencing strategies

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 .

Ordered Clone approach

Large DNA fragment Whole Genome Randomly sequence fragments Fill gaps Repeat for entire genome map

Digest and subclone

Random sequencing approach

Whole Genome

Shear and subclone Randomly sequence fragments Fill gaps

Assembly Phase
The assembly phase is composed of three major steps :
The conversion of the data from automated sequencers to

nucleotide sequences. The utilization of these sequences in the assembly process. The continuous assessment of this assembly process.

Phred

Automated Sequencers

Phred
Phred is a base-calling program for DNA sequence traces. Phred reads DNA sequence chromatogram files and analyzes the peaks to call bases, assigning ("Phred scores") to each base call.

Sequence assembly and Gap closure

Random sequences initially interpreted using highly accurate base calling software and assembled to generate primary contigs** using software such as Phrap. Computational and experimental techniques used to identify linking clones and order primary contigs** Primer walk sequencing of linking clones and PCR products to fill sequence gaps between contigs** Confirmation of contig** order by PCR

**The DNA sequence reconstructed from a set of overlapping DNA segments

Linking Clones
Contig 1
FWD

Large Insert Linking Clone

Contig 2
REV

Primer Walking** 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.

Physical Gaps
Peptides can link the

coting ends having regions with homology to the same gene (or operon / gene cluster) Physical gaps can also be filled with the help of Southern Hybridization.
Linked by Southern Hybridization

Contig 2
FWD

PCR Product

Contig 6
REV

Annotation
Sequence Gene prediction Proteins Similarity searches against reference databases Calculations & predictions (MW , structure, location etc)

Annotated Proteins Pathway prediction Annotated Proteins & pathways Manual editing Data Generation

Completely Sequenced Microbial Genome

Over View

Conclusion
Microbial genome sequencing and analysis is a rapidly expanding and increasingly important strand of microbiology, Important information about the specific adaptations and evolution of a microorganism can be determined from genome sequencing. However, genome sequencing merely a strong starting point on road to completely understand the biology of microorganisms

References
Lionel .F., Etal .,(1999) .Cloning and assembly strategies in

microbial genome projects. Microbiology Res145, 26252634. Claire M. F., Jonathan A. E. and Steven L. S.,(2000).Microbial genome 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.

Presented by,

Devarapalli Pratap