microbial genomics
DESCRIPTION
Microbial Genomics. Topics Describe the new area of genomics Outline the rapid progress in genomic sequencing Describe the analysis of sequences - bioinformatics Show the use of genomics in the study of microbes - PowerPoint PPT PresentationTRANSCRIPT
Dr M. D-S, 2007
Microbial GenomicsMicrobial GenomicsMicrobial GenomicsMicrobial Genomics
Topics Describe the new area of genomics Outline the rapid progress in genomic sequencing Describe the analysis of sequences - bioinformatics Show the use of genomics in the study of microbes Use the sequence of a human pathogen Escherichia
coli O157:H7 to illustrate the above points Ref: Perna et al. (2001) Nature 409:529 (USA)
Relevant to next lectures.
Dr M. D-S, 2007
Microbial genome sequencesMicrobial genome sequencesMicrobial genome sequencesMicrobial genome sequences
Genbank (NCBI), Bethesda, Maryland, USA 2007: 481 - completed microbial genomes 2006: 3192003: 112
Sizes range from 0.58 - over 9 Mb
Genbank - main genomic database
There is some duplication...
Dr M. D-S, 2007
GenomicsGenomicsGenomicsGenomics- the study of entire genomes of organisms
assumes the entire sequence of at least one representative example has been determined
includes study of all the genes and gene products and non-coding regions
includes study of genome organisation and evolution
- the study of entire genomes of organismsassumes the entire sequence of at least one
representative example has been determined includes study of all the genes and gene
products and non-coding regions includes study of genome organisation and
evolution
Dr M. D-S, 2007
The The explosionexplosion of of ‘‘-ome’-ome’ and ‘ and ‘-omics’-omics’ words words
The The explosionexplosion of of ‘‘-ome’-ome’ and ‘ and ‘-omics’-omics’ words words
Functional genomicsProteomeTranscriptomeMetabolome, Glycome, Lipidome
e.g. a recent journal article with the title: “Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis”
Functional genomicsProteomeTranscriptomeMetabolome, Glycome, Lipidome
e.g. a recent journal article with the title: “Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis”
Dr M. D-S, 2007
What can microbial genomics tell us ?• Full gene complement of the cell
• Complete description of cell metabolism
• How genomes are structured
• Virulence genes
• Potential drug targets
• Gene flow between cells (evolution)
GenomicsGenomicsGenomicsGenomics
Dr M. D-S, 2007
Genome Sequencing:Genome Sequencing:TwoTwo methodsmethods
Genome Sequencing:Genome Sequencing:TwoTwo methodsmethods
1. Sanger di-deoxy sequencing (using fluorescently labelled ddNTPs) on cloned DNA templates.
2. Pyro-sequencing method on 454 machine using uncloned DNA templates
1. Sanger di-deoxy sequencing (using fluorescently labelled ddNTPs) on cloned DNA templates.
2. Pyro-sequencing method on 454 machine using uncloned DNA templates
Dr M. D-S, 2007
Genome Sequencing:Genome Sequencing:TwoTwo methodsmethods
Genome Sequencing:Genome Sequencing:TwoTwo methodsmethods
Dye-terminator chemistry, ABI sequencing apparatus, commercial software for handling seq. data
1. Sanger di-deoxy sequencing (using fluorescently labelled ddNTPs) on cloned DNA templates. ‘Shotgun’ strategy.
Dr M. D-S, 2007
Genomic sequencing Genomic sequencing methodsmethods
Genomic sequencing Genomic sequencing methodsmethods
Shear DNA & isolate Shear DNA & isolate fragments about 2kbfragments about 2kb
chDNA
Clone thousands of Clone thousands of fragments into fragments into plasmid vector plasmid vector (library). Prepare (library). Prepare DNA for DNA for sequencingsequencing
Dr M. D-S, 2007
Dideoxy chain terminationDideoxy chain terminationDideoxy chain terminationDideoxy chain termination
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
http://www.plattsburgh.edu/acadvp/artsci/biology/bio401/DNASeq.html
Dr M. D-S, 2007
Sequence: methods sectionSequence: methods sectionSequence: methods sectionSequence: methods sectionApplied Biosystems Inc (ABI) latest
sequencing machine, PE 3700
Capillary electrophoresis
96 capillaries at a time
Robotically loaded and run (24hr)
How many bp can it do in a day??
- each run is 2hr, get 600-1000 nt per capillary, 96 capillaries/run
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Dr M. D-S, 2007
Applied Biosystems Inc (ABI) latest sequencing machine, PE 3700
How many bp can it do in a day??
- each run is 2hr, about 800bp each lane, 96 lanes
= 24/2 800 96 = 921,000
Or about 1 Mb /machine/day
Sequence: methods sectionSequence: methods sectionSequence: methods sectionSequence: methods section
Dr M. D-S, 2007
Sequence dataSequence dataSequence dataSequence dataLaser scanning of the 96 Laser scanning of the 96 capillary tubes identifies the capillary tubes identifies the colour and positions of the colour and positions of the closely spaced bands of closely spaced bands of ssDNA.ssDNA.
Top of capillary tubes
TAATCATGGTC....TAATCATGGTC....
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Dr M. D-S, 2007
~ 1 Mb /machine/day
Shotgun sequencing: how much do Shotgun sequencing: how much do you need to do?you need to do?
Shotgun sequencing: how much do Shotgun sequencing: how much do you need to do?you need to do?
Want both strands, good sequence for both, random coverage means you will need 6-8x genome size in sequence data
Speed makes it efficient?
Counter argument is the difficulty in linking up reads, particularly when genomes have long repeat sequences.
Dr M. D-S, 2007
Genome Sequencing:Genome Sequencing:TwoTwo methodsmethods
Genome Sequencing:Genome Sequencing:TwoTwo methodsmethods
In the E.coli O157:H7 genome sequence paper by Perna et al., there were 2 gaps remaining in the genome sequence! They couldn’t complete it.
“Extended exact matches pose a significant assembly problem.” ??
Dr M. D-S, 2007
Repeat sequences, eg. Prophage genomesRepeat sequences, eg. Prophage genomesRepeat sequences, eg. Prophage genomesRepeat sequences, eg. Prophage genomes
Nearly identical prophage sequences at 3 locations on genome, all > 2000 nt
Nearly identical prophage sequences at 3 locations on genome, all > 2000 nt
What sequences do you observe
when inside a prophage genome?
What sequences do you observe
when inside a prophage genome?
Dr M. D-S, 2007
Repeat sequences, eg. Prophage genomesRepeat sequences, eg. Prophage genomesRepeat sequences, eg. Prophage genomesRepeat sequences, eg. Prophage genomes
Nearly identical prophage sequences at 2 locations on genome
Nearly identical prophage sequences at 2 locations on genome
What sequences do you see going
across the borders of prophages?
What sequences do you see going
across the borders of prophages?
Dr M. D-S, 2007
Repeat sequences, eg. Prophage genomesRepeat sequences, eg. Prophage genomesRepeat sequences, eg. Prophage genomesRepeat sequences, eg. Prophage genomes
Nearly identical prophage sequences at 2 locations on genome
Nearly identical prophage sequences at 2 locations on genome
What information do you need to
place the repeats properly?
What information do you need to
place the repeats properly?
Dr M. D-S, 2007
Genome Sequencing:Genome Sequencing:TwoTwo methodsmethods
Genome Sequencing:Genome Sequencing:TwoTwo methodsmethods
1. Sanger di-deoxy sequencing (using fluorescently labelled ddNTPs) on cloned DNA templates.
2. Pyro-sequencing method on 454 machine using uncloned DNA templates
1. Sanger di-deoxy sequencing (using fluorescently labelled ddNTPs) on cloned DNA templates.
2. Pyro-sequencing method on 454 machine using uncloned DNA templates
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
The 454 machines: the next revolution
www.454.com
The 454 machines: the next revolution
www.454.com
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
40 million bases/5.5 hr
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
The 454 machines: the next revolution
www.454.com
40 million bases/5.5 hr
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
DNA immobilised on micro-beads
Positioned in wells of special tray (44um diameter, 1.2 million per chip)
Sequencing enzymes on smaller beads.
Only one DNA-bead can fit in each well
Each bead has only one DNA fragment attached, so will give unique sequence.
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
The 454 machines: the next revolution
www.454.com
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
When a base is incorporated (by DNA polymerase), light is emitted, and the light detected under each well.
The 454 machines: the next revolution
www.454.com
40 million bases/5.5 hr
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
When a base is incorporated (by DNA polymerase), light is emitted, and the light detected under each well. If there are multiple bases, the light is proportional to the number. Chain lengths of 200 nt are possible. With 200,000 wells, and 200nt/well, then 40 million bases can be sequenced.
Dr M. D-S, 2007
Papers filled with JARGON. Mainly genetic terms. Some terms are relatively new (eg. replichore)
Use the E.coli paper example, stopping to investigate each new term or concept
Emphasise the uses of this data, and the future of genomic research.
GenomicsGenomicsGenomicsGenomics
Dr M. D-S, 2007
What do you know about What do you know about microbial genomes ?microbial genomes ?
What do you know about What do you know about microbial genomes ?microbial genomes ?
Exercise: Think of a typical bacterial genome, like that of E.coli and -Sketch the genome and the most significant
features you know about it (as a whole genome, not individual genes)
Jot down what you think the main selective pressures are on it
Exercise: Think of a typical bacterial genome, like that of E.coli and -Sketch the genome and the most significant
features you know about it (as a whole genome, not individual genes)
Jot down what you think the main selective pressures are on it
Dr M. D-S, 2007
Escherichia coliEscherichia coli genome genomeEscherichia coliEscherichia coli genome genome
Circular, ~ 4.6 MbOri and Ter, bidirectional replicationReplichores about equal
Circular, ~ 4.6 MbOri and Ter, bidirectional replicationReplichores about equal
ter
oriC
Dr M. D-S, 2007
Replichore ‘balance’Replichore ‘balance’ ? ?Replichore ‘balance’Replichore ‘balance’ ? ? If you move oriC relative to Ter, the
growth rate of E. coli K-12 is reduced.Chromosomal inversions around the origin
or termination of replication are usually symmetrical, conserving the replichore balance.
Hill, C. W., and J. A. Gray. 1988. Effects of chromosomal inversion on cell fitness in Escherichia coli K-12. Genetics 119:771–778.
Eisen, J. A., J. F. Heidelberg, O. White, and S. L. Salszberg. 2000. Evidence for symmetric chromosomal inversions around the replication origin in bacteria. Genome Biol. 1:0011.1–0011.9
If you move oriC relative to Ter, the growth rate of E. coli K-12 is reduced.
Chromosomal inversions around the origin or termination of replication are usually symmetrical, conserving the replichore balance.
Hill, C. W., and J. A. Gray. 1988. Effects of chromosomal inversion on cell fitness in Escherichia coli K-12. Genetics 119:771–778.
Eisen, J. A., J. F. Heidelberg, O. White, and S. L. Salszberg. 2000. Evidence for symmetric chromosomal inversions around the replication origin in bacteria. Genome Biol. 1:0011.1–0011.9
Dr M. D-S, 2007
E.coliE.coli genome - global features genome - global featuresE.coliE.coli genome - global features genome - global features
Gene dosageGene direction relative to oriRecombination/inversion rates vary
around chromosome
Gene dosageGene direction relative to oriRecombination/inversion rates vary
around chromosome
Dr M. D-S, 2007
Gene DosageGene DosageGene DosageGene Dosage Genes near the origin of replication will
almost always be in multiple copy compared to genes near the terminus
So the position of a gene relative to the origin will affect its expression, and the regulatory systems would have evolved to accommodate for the gene dosage effect.
So what would happenif you moved genes ?
Genes near the origin of replication will almost always be in multiple copy compared to genes near the terminus
So the position of a gene relative to the origin will affect its expression, and the regulatory systems would have evolved to accommodate for the gene dosage effect.
So what would happenif you moved genes ?
ter
oriC
Dr M. D-S, 2007
Gene DirectionGene DirectionGene DirectionGene Direction
What happens when a DNA pol meets an RNA pol going in the opposite direction?
What happens when a DNA pol meets an RNA pol going in the opposite direction?
DNAPolymerase
RNAPolymerase
Dr M. D-S, 2007
Gene DirectionGene DirectionGene DirectionGene Direction
What happens when a DNA pol meets an RNA pol going in the opposite direction?
What happens when a DNA pol meets an RNA pol going in the opposite direction?
This is better….DNA
Polymerase
RNAPolymerase
Dr M. D-S, 2007
Gene DirectionGene DirectionGene DirectionGene Directionori
A preference for genes to be on ONE strand of the replichore, so that the direction of transcription and replication are the same.
This bias may have other implications.
Dr M. D-S, 2007
Recombination/inversionsRecombination/inversionsRecombination/inversionsRecombination/inversions
Genomes often have large repeated sequences, eg. ribosomal RNA gene clusters (16S-23S-5S), or phage genomes.
Such repeats allow large inversions of DNA segments or recombination between chromosomes
Genomes often have large repeated sequences, eg. ribosomal RNA gene clusters (16S-23S-5S), or phage genomes.
Such repeats allow large inversions of DNA segments or recombination between chromosomes
Dr M. D-S, 2007
Inversion via repeated Inversion via repeated sequencessequences
Inversion via repeated Inversion via repeated sequencessequences
Homologous recombination between rRNA genes
Homologous recombination between rRNA genes
Dr M. D-S, 2007
Systematic bias in base composition of one strand as you go around the genome
GC skew
genome
originorigin
terterterter
[G-C]
[G+C]
Genomics: What is GC-skew ?Genomics: What is GC-skew ?Genomics: What is GC-skew ?Genomics: What is GC-skew ?
Dr M. D-S, 2007
GC-skew of genomic DNA
Compositional bias:
Leading strand enriched in G/T (keto)
Lagging strand enriched in C/A (amino)
WHY?
Perhaps due to deamination of exposed C’s in the leading strand, producing C>T mutations. Theory only.
Dr M. D-S, 2007
Chi sequencesGCTGGTGG
Sequence recognised (and cut) by the RecBC enzyme
Promotes homologous recombination (by RecA)
E.coliE.coli O157:H7O157:H7 -- K12K12 genome genome comparison:comparison:
E.coliE.coli O157:H7O157:H7 -- K12K12 genome genome comparison:comparison:
Dr M. D-S, 2007
Lateral Gene Transfer (LGT)Lateral Gene Transfer (LGT)Lateral Gene Transfer (LGT)Lateral Gene Transfer (LGT) Literally, the natural transfer of genetic material
between different organisms (species, genera, etc) Doesn’t say how the DNA was transferred or
integrated, or where it came from. Does imply that the DNA can be identified as
‘foreign’ Since DNA doesn’t have a ‘made in X’ sticker,
how can the ‘foreignness’ be identified? …. Ideas?….
Dr M. D-S, 2007
Lateral Gene TransferLateral Gene Transfer (LGT) (LGT)Lateral Gene TransferLateral Gene Transfer (LGT) (LGT)Known mechanisms of DNA transfer between
bacteria:- Transduction
transducing bacteriophages introduce host DNA, and this recombines with the genome
Transformation DNA uptake from the surroundings, and
recombination. Conjugation
natural transfer method, sex pilus, one-way transfer, recombination.
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Dr M. D-S, 2007
ProphageProphageProphageProphage
The host cell, with a prophage, is called a lysogen. Some prophages express virulence determinants,
such as toxins ( = lysogenic conversion). eg. Shiga toxin
Some prophages exist as plasmids, but most integrate into the genome.
If the prophage becomes damaged…. ?
Bacteriophages that are temperate (as compared to Bacteriophages that are temperate (as compared to lytic) can exist inside host cells in a stable and lytic) can exist inside host cells in a stable and relatively inactive state as prophages.relatively inactive state as prophages.
Dr M. D-S, 2007
E.coliE.coli genome sequences genome sequencesE.coliE.coli genome sequences genome sequencesSTRAIN SIZE DATEE.coli K12 4639221 bp, Oct 13 1998
E.coli O157:H7 (USA)5528970 bp, Jan 25, 2001
E.coli O157:H7 (Japanese) 5498450 bp, Mar 7, 2001
*about 4.1Mb in common
Data from NCBI:
http://www.ncbi.nlm.nih.gov:80/PMGifs/Genomes/eub.html
Dr M. D-S, 2007
E.coliE.coli O157:H7 - K12 O157:H7 - K12 genome comparisongenome comparisonE.coliE.coli O157:H7 - K12 O157:H7 - K12 genome comparisongenome comparison
Unexpected complex segmented relationship Share a common 4.1 Mb ‘backbone’ or common,
and generally colinear sequence (only 1 inversion) Homologous sequences are interspersed with
HUNDREDS of ISLANDS of INTROGRESSED DNA
A B C D
A B C D
A B X C D
A B C D
Dr M. D-S, 2007
The specific DNA segments for each strain were named ‘O islands’ , ie O157:H7-specific DNA segments, or ‘K islands’
Backbone of 4.1 Mb common sequence. Not identical (eg 75% of proteins differ by one aa).
O-islands total 1.34 Mb (about 26% of genes !) Largest O-island is 106 gene region (not small!)
E.coliE.coli O157:H7 - K12 O157:H7 - K12 genome comparisongenome comparisonE.coliE.coli O157:H7 - K12 O157:H7 - K12 genome comparisongenome comparison
Dr M. D-S, 2007
Virulence genes do not seem to be concentrated in one particular ‘island’; appear to be several
Often (189 cases), the backbone-island junction is WITHIN an ORF.
Protein coding ORF
AUG UGAO-islandO-island
What does this pattern suggest?What does this pattern suggest?
E.coliE.coli O157:H7 - K12 O157:H7 - K12 genome comparisongenome comparisonE.coliE.coli O157:H7 - K12 O157:H7 - K12 genome comparisongenome comparison
Dr M. D-S, 2007
Suggests that incoming DNA recombined with the genome (somehow?) rather than inserted.
E.coliE.coli O157:H7 - K12 O157:H7 - K12 genome comparisongenome comparisonE.coliE.coli O157:H7 - K12 O157:H7 - K12 genome comparisongenome comparison
Protein coding ORF
AUG UGAO-islandO-island
Dr M. D-S, 2007
Distribution of O-islands of EDL933 specific sequence (red), ‘K-islands’ of K12 specific sequence (green) and common ‘backbone’ sequence (blue)
GC-content of genes, plotted around mean
GC-skew for 3rd codons
Scale, in base pairs
Octamer Chi sequences
Genome Map
Dr M. D-S, 2007
Genome sequence - Figure 2Genome sequence - Figure 2Genome sequence - Figure 2Genome sequence - Figure 2O-specificO-specific ‘islands’
K-specificK-specific ‘islands’
Scale (10kb/tick)
O157:H7 genes and their orientation
Dr M. D-S, 2007
Genome sequence - Figure Genome sequence - Figure 22
Genome sequence - Figure Genome sequence - Figure 22
CP-933 = Cryptic Prophage. Also an O island
How many kb is this phage genome?
Dr M. D-S, 2007
Summary of main findings:
1. Many insertions of DNA around chromosome
2. Inserted DNA is foreign (HGT or Lateral GT)
3. Several virulence gene clusters; widely spread
4. Prophage genomes prominent
5. Systematic variations base composition- coding strand, GC skew, chi seqs
E.coliE.coli O157:H7O157:H7 genome sequence genome sequenceE.coliE.coli O157:H7O157:H7 genome sequence genome sequence
Dr M. D-S, 2007
Summary of main findings:
6. E.coli O157:H7 undergoes relatively high rates of recombination and mutation.
- where is the DNA coming from ? unknown, phage, mobile elements (eg. transposons)
- what is the main method of transfer ?
- is defective DNA mismatch repair important ?
E.coliE.coli O157:H7O157:H7 genome sequence genome sequenceE.coliE.coli O157:H7O157:H7 genome sequence genome sequence
Dr M. D-S, 2007
Summary of main findings:These large differences can be exploited: Diagnostic tools (discriminate b/n E.coli strains) New virulence gene candidates can be tested for
function, and new drugs developed Effects of antibiotics on toxin synthesis examined
Note in the genome sequences of many microbes, the percentage of ORFs that cannot be identified is often > 20%
E.coliE.coli O157:H7O157:H7 genome sequence genome sequenceE.coliE.coli O157:H7O157:H7 genome sequence genome sequence