1. WHOLE GENOME SEQUENCING OF
BACTERIA & ANALYSIS
ELAMURUGAN. A
Ph.D Scholar,
Vet. Immunology

2. INTRODUCTION
 1977 - first complete genome to be sequenced was
bacteriophage X174 - 5386 bp
 1995 - first complete genome sequence from a free living
organism - Haemophilus influenzae (1.83 Mb) by whole
genome shotgun approach
 Sanger & Coulson (1977) - used chain-terminating
dideoxynucleotide analogues
 Maxam & Gilbert (1977) chemical degradation DNA
sequencing - terminally labeled DNA fragments were
chemically cleaved at specific bases and separated by gel
electrophoresis

3. http://www.genomesonline.org/cgi-bin/GOLD/sequencing_status_distribution.cgi
429
Genome online database (GOLD)

4. ARCHON X PRIZE
 X PRIZE Foundation in Santa Monica, CA, has
introduced the Archon X PRIZE for Genomics and will
award a sum of $10 million to the first team that can
design a system capable of sequencing 100 human
genomes in 10 days

5. SEQUENCING TECHNOLOGY
 First generation
 Sanger’s dideoxy chain terminating tech
 Maxam & Gilbert chemical degradation tech
 Next generation sequencing (NGS)
 454/Roche - pyrosequencing
 Illumina/ Solexa - reversible dye terminators
 SOLiD /ABI- sequential ligation of oligonucleotide probes
Second generation HT-NGS – sequencing after amplification

7.  Heliscope
 SMRT (Pacific biosciences)
 Single molecule real time (RNAP) sequencer
 Nanopore DNA sequencer
 Ion Torrent sequencing technology (PostLight)
 VisiGen biotechnologies – FRET
 Advantages of 3rd generation HT-NGS over 2nd
 higher throughput
 faster turnaround time
 longer read lengths
 higher consensus accuracy
 small amounts of starting material
 low cost
Third
generation
HT-NGS -
Single
molecule
sequencing

9. ADVANTAGES OF HT-NGS
 Massive parallel sequencing of hundreds of thousands
or millions of templates
 Preliminary and tedious cloning work is eliminated and
substituted by PCR amplification
 Most recent technologies, even PCR is eliminated,
because single DNA molecules
 Economic
 Reduced time

10. DISADVANTAGES OF HT-NGS
 Most NGSTs produce short reads
 Constructions of fragment libraries remain tricky and
involve several steps of fragmentation, adaptor ligation
and PCR amplification
 Short homopolymers with the 454 technology
 Modified nucleotides cause mis-incorporation or block
further incorporation if the florescent moiety cannot be
completely removed
 Assembly of short reads into longer sequences

12. Illumina/ Solexa technology

16. zero-mode
waveguides
(ZMWs)

17. Selection of a technology for an experiment

18. GENOME ASSEMBLY
 Assemblers can join sequences together based on
overlapping regions between the sequences
 Composed of contigs and scaffolds
 Contigs - contiguous consensus sequences that are
derived from collections of overlapping reads
 Scaffolds - ordered and orientated sets of contigs that are
linked to one another by mate pairs of sequencing reads
 N50 - basic statistic for describing the contiguity of a
genome assembly. The longer the N50 is, the better the
assembly

19.  Alignment against a reference genome sequence
 De novo assembly Construction of longer sequences, such
as contigs or genomes, from shorter sequences, such as
sequence reads, without prior knowledge of the order of
the reads or reference to a closely related sequence

20. GENE PREDICTION
 Ab initio gene prediction - mathematical models
rather than external evidence (such as EST and
protein alignments) to identify genes and to
determine their intron–exon structures
 Evidence-driven gene prediction - using ESTs, can
be used to identify exon boundaries
unambiguously. Great potential to improve the
quality of gene prediction in newly sequenced
genomes. ESTs and proteins must first be aligned
to the genome
 Commonly used tools for gene prediction in
prokaryotes Glimmer, GeneMark

21. GENOME ANNOTATION
 Is the extraction of biological knowledge from raw
nucleotide sequences
 Seeks to identify every potential protein coding gene
(ORFs)
 Used to compare in available database like BlastP
 ‘Structural’ genome annotation is the process of identifying
genes and their intron–exon structures
 ‘Functional’ genome annotation is the process of attaching
meta-data such as gene ontology terms to structural
annotations

24. APPLICATIONS
 Very large no of short reads help to identify single nucleotide
polymorphisms (SNP) when comparing them in reference
genome
 Identification of rearrangements, deletions, insertions,
inversions
 Used to generate expressed sequence tags (EST) from RNA
sequencing
 Also to detect small regulatory RNAs
 Illumia technoloy - ChIP Seq to study protein - DNA
interactions
 Metagenomics

25. LEADS TO DEVELOPMENT
 Functional genomics
 Comparative genomics
 Environmental genomics (Metagenomics)

26. FUNCTIONAL GENOMICS
 Reveals genome structure and its functional relation
 Orthologs - they represent genes derived from a common
ancestor that diverged because of divergence of the
organism, tend to have similar function
 Paralogs are homologs produced by gene duplication and
represent genes derived from a common ancestral gene
that duplicated within an organism and then diverged, tend
to have different functions
 Xenologs are homologs resulting from the horizontal
transfer of a gene between two organisms. The function of
xenologs can be variable, depending on how significant the
change in context was for the horizontally moving gene. In
general, though, the function tends to be similar

27. PHYLOGENETIC ANALYSIS
 Phylogenetic trees, which are used to classify the
evolutionary relationships between homologous
genes represented in the genomes of divergent species
Internal Nodes or
Divergence Points
Branches or
Lineages A
B
C
D
E
Terminal Nodes
Ancestral Node
or ROOT of
the Tree

28. COMPARATIVE GENOMICS
 Comparison of genome sequences reveals much
information about genome structure and evolution,
including importance of lateral gene transfer
 Tool to discover how microbs adapted to particular
ecology and in development of new therapeutic
agents

29. METAGENOMICS
 Genomics-based study of genetic material
recovered directly from environmentally derived
samples without laboratory culture and compared
with all previously sequenced genes
 Enable how microbs adapt extreme environments
which help to discover new metabolic pathway and
protective mechanisms

30. IMPACT OF GENOME SEQUENCING
 Revealed genome reduction in I/C bacteria
 Genome plasticity (rearrangements, mobile elements)
 Gene duplication and diversification of protein function
 Lateral gene transfer & acquisition of new functions
 Adaptation to environments, virulence
 Industrial process - fermentation tech,
 Bioremediation
 Biotransformation
 Development of vaccines
 Bacterial diversity
 Synthetic biology
 Epigenetics

31. REVERSE VACCINOLOGY
 Use of genomic sequence information to identify novel
and better suited protein candidates for vaccine
 Serogroup B Neisseria meningitidis – based on
genomic data all proteins predicted to be surface
exposed, therefore accessible to antiobodies
 Suitable candidates selected after sequencing various
strains
 Streptococcus agalactiae
 Pan-genome composed of core genome, the genes
present in all sequence strains and the dispensable
genome made of genes present in a subset of strains

32.  Synthetic biology - from sequence of entire genome to
synthesize genes de novo
 Identification of minimal genome, the smallest set of
genes that enbles life - Mycoplasma genitalium

33. DATABASES AND TOOLS RELATED WITH BACTERIAL
GENOMIC DATA
 NCBI Entrez Genome Project database:
 http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db = genomeprj
 A searchable collection of complete and incomplete (in-progress)
large-scale sequencing, assembly, annotation, and mapping projects
for cellular organisms
 NCBI, Bacteria Genome Database:
 http://www.ncbi.nlm.nih.gov/genomes/static/eub.html
 The Genome database provides views for a variety of genomes,
complete chromosomes, sequence maps with contigs, and integrated
genetic and physical maps
 Bacterial Genomes at The Sanger Institute:
• http://www.sanger.ac.uk/Projects/Microbes/
• This web contains a list of funded, on-going, or completed projects of
pathogens sequenced at this institute
 TIGR Comprehensive Microbial Resource (CMR):
 http://cmr.tigr.org/tigr-scripts/CMR/CmrHomePage.cgi
 A free website displaying information on all the publicly available,
complete prokaryotic genomes

34.  GOLD: Genomes OnLine Database:
 http://www.genomesonline.org/
 A genome database containing information about which genomes have
been sequenced or are in progress
 Microbial Genome Database for Comparative Analysis (MBGD):
 http://mbgd.genome.ad.jp/
 A database for comparative analysis of completely sequenced microbial
genomes
 Virulence Factors of Bacterial Pathogens (VFDB):
 http://zdsys.chgb.org.cn/VFs/main.htm
 VFDB is an integrated and comprehensive database of virulence
factors for bacterial pathogens
 Genome Information Broker:
 http://gib.genes.nig.ac.jp/
 A comprehensive data repository of complete microbial genomes in the
public domain. Many microbial genomes can be explored graphically
 Islander, a Database of Genomic Islands:
 http://www.indiana.edu/~islander
 This database contains genomic islands discovered in completely
sequenced bacterial genomes

35.  GenoList genome browser at Institute Pasteur:
 http://genolist.pasteur.fr/
 Contains access to diverse genome browsers of pathogenic
bacteria
 IslandPath:
 http://www.pathogenomics.sfu.ca/islandpath/update/IPindex.pl
 An aid to the identification of genomic islands, including
pathogenicity islands, of potentially horizontally transferred genes
 HGT-DB:
 http://www.tinet.org/~debb/HGT/
 A database containing the prediction of horizontally transferred
genes in several prokaryotic complete genomes
 E. coli genome project:
 http://www.genome.wisc.edu
 A site devoted to the E. coli genome project with an updated
annotation of the genome