Week 1

genetic variation in a population

generated by mutation

variants

passed to offspring by vertical transmission or between individuals by horizontal gene flow

selection

favourable variants shapes which members of the population survive

organisms occupying the same niche

• they are in necessarily in competition

• physical niches i..e gut or soil

• geographic/demographic niches i.e australian soils or guts of children

• nutritional niches i.e lactose fermenters or phototrophs

• competition means two organisms cannot occupy the same niche for long — have to evolve different niches or one will out compete the other and drive it to extinction

vertical transmission

direct replication of the chromosome during cell division

• if replicaiton is faithful → both daughter cells are genetically identical

• if errors in replication → new genetic variants will rise and be in competition with other variants of their species

random mutation

random changes in gnetic code due to DNA damage or replication errors.

• increased diversity (relatively slow)

horizontal gene transfer

Movement of genetic material from one organism to another independent of cell division

• increased diversity (rapid)

genetic drift

random changes to the frequency of variants through mutation or HGT

• may increase/decrease (tends to impact loci under weak selective pressure more strongly)

selection

increase survival of most fit variants

• decreased diversity (impact depends on strength of selection)

migration

Movement of subset of the population into a new isolated region (or host population), generates a genetic bottleneck

• decreased diversity (rapid, often heavily random)

allele

each variant (gene/promoter/other element) differing by one or more nucleotides — normally alleles all share the same function

bacterial species

collection of strains with a conserved core of genes and phenotypes

strain

subvariant of a bacterial species with a common ancestor

• may be defined by genetic content and/or phenotype

• strains of the same species may have very different phenotypes

isolates

individual pure cultures from different sources

• represent a single snapshot of an evolving lineage (and can themselves evolve in the lab)

clade

• group consisting of all organisms descended from a single common ancestor

• may include multiple species or genera but is typically used in bacteriology to refer to groupings within a species

clone

group of genetically homogenous cells which have arisen from a single parent cell

clonality

tendency of a population towards forming clonal groups

• more clonal organisms tend to mostly evolve slowly through mutations and binary cell division rather than rapidly evolving and acquiring new genes

horizontal gene transfer

results in acquistion of genetic material from outside the cell

• may occur through recombination of foreign DNA into the chromosome

• by movement of mobile genetic elements i.e plasmids, phages, integrative and conjugative elements

• “two roads to rome” of bacterial evolution

spotaneous DNA damage

• error in DNA replication is the main cause

• rate of 1 in 10⁸ to 10¹¹ nucleotides is copied incorrectly by the DNA polymerase which uses proof reading activity to correctly copy the template

induced DNA damage by chemical alteration

• alkylation

• UV-induced thymine dimers

• oxygen radicals

• initial damage, if not repaired, will result in muation of the DNA which will be in herited by daughter cells via cell division

alkylation

• electrophiles add alkyl groups to phosphates, stalls replication

• carcinogens, ethylmethane sulphonate

UV-induced thymine dimers

• DNA absorbs UV at 260nM

• forms intra-strand pyrimidine dimers, main T-T

• distortion of double helix prevents DNA replication → lethal

oxygen radicals

• cause single and double stranded breaks

• gamma radiation and x-ray

direct repair mechanisms

• restoration to original undamaged state

• photoreactivation

• nucleotide excision repair (short match repair)

• mismatch repair

indirect repair

• damage bypass system using DNA replication (not necessarily restoring the undamaged state)

• recombination repair

• SOS repair

silent mutation

• no effect on protein sequence

• due to degeneracy of genetic code

missense mutation

one amino acid in the protein is replaced by another

• replacement with amino acid of similar biochemical profile

• replacement with an amino accid with a differnt biochemical profile

• can result in complete or partial loss of function, change in function or temperature sensitive mutants

nonsense mutation

mutation gives rise to stop codon

• stop codons TAG (amber), TGA (opal), TAA (ochre)

• usually results in complete loss of function as rpemature termination of polypeptide chain gives truncated protein

In-frame indel

loss or gain of a multiple of 3 bases results in insertion or deletion of amino acids

• often does not result in complete loss of function

frame shift mutation

• insertion shift DNA sequence out of frame

• usually results in complete loss of function — either premature stop codon or entirely new amino acid sequence

• impact depnds on location within the gene

mutation rate is impacted by

• genetic drift

• population size — larger populations have slower rates of drift

• ecology — some lifestyles select for rapid mutation e.g. host restricted bacteria/viruses undergoing rapid transmission face frequent selection bottlenecks

dN/dS ratio

• degree of selection acting on a gene is given by this

• dN = non-synonymous nucleotide substitutions per non-synonymous site

• dS = synonymous nucleotide substitution per synonymous site

intepreting dN/dS

• non-synonymous mutations are presumed to impact fitness — expect them less frequently in residues under greater selective pressure

• dN/dS=1 there is no selective pressure (neutral selection)

• dN/dS<1 deleterious alleles are rapidly removed from the population (purifying sleection, the locos is conserved)

• dN/dS>1 polymorphisms are frequent and are retained in the population (positive selection, the locus is flexible)

homologs

• pair of two sequences which are predicted to have a common ancestor

• can arise through gene duplication (makes paralogs) or speciation (produces orthologs)

assumptions of models of homology

• identical residue pairs are aligned

• different amino acids with similar physiochemical properties will form a pair

• scores are attributed to each possible matches, mismatch and gap

• gaps represent indels in an evolutionary context

• highest total score is most likely relationship

measuring homology of nucleotides

• either a base matches or it doesn’t

• can measure the percentage of nucleotide matching in an alignment to measure the homology — % identity

• can be interpreted as evolutionary distance between sequences

epidemiology

study of distribution and determinants of disease and health related events and its application in control and prevention

epidemiology

• deals with one defined population at risk

• risks lead directly to cases

• identifies previously unknown causes

infectious disease epidemiology

• two or more populations (human, pathogen, possible vectors or alternate hosts)

• a case is itself risk factor

• the cause often known

two or more populations

• humans — sometimes the only reservoir of pathogens

• infectious agents — helminths, bacteria, fungi, protozoa, virusese, prions

• vectors — mosquito, snails, blackfly

• animals — sheep, goats, mice and ticks, bats

what is infectious disease epidemiology used for?

• identification of causes of new, emerging infections

• surveillance of infectious disease

• indentification of source of outbreaks (environmental or human)

• studies of routes of transmission and natural history of infections

• identification of new interventions

R

• the basic reproductive number — R0

• the mean number of individuals directly infected by an infectious case through the total infectious period, when introduced to a susceptible population

• Endemic (R=1) — transmission occurs but numbers of cases remains constant

• Epidemic (R>1) — the number of cases increases

• Pandemic (R>1) — when epidemics occur at several continents (global epidemic)

identification of the infectious agent is required

• to administer the treatment

• for prognosis

• to initiate appropriate infectious disease control measures

• to take suitable preventive steps

• to understand epidemiology

• to know the disease history

phenotypic microbial typing methods

• based on the assumption that an organims genotype affects its phenotype — therefore the phenotype tells us about the relatedness of organisms

• rapid and inexpensive

• i.e biotyping, antibiotyping, serotyping, phage-typing

API strips

• grow bacteria from patient sample (overnight), inoculate API strip and incubate

• capsules contain various tests for metabolic activities which give colorimetric readings

• 15 identification systems covering more than 600 bacteria species

time drawbacks of phenotyping

• does not detect emergence of noval variants

• culture dependent which makes it slow for some organisms

• requires more infrastructure than molecular typing

sensitivity drawbacks of phenotyping

• antimicrobial treatment iff often commenced before sampling for diagnosis — microbe dies and samples are not useful for phenotyping (almost 50% of diagnostic samples are in this category)

• some diseases have very low infectious doses and are difficult to detect

accuracy drawbacks of phenotyping

• seropositivity may be confounded by cross reaction of antibodies to commensals or other pathogens

• antibiotyping may be confounded if lab conditions do not result in the expression of antimicrobial resistance (AMR) determinants

• phage typing can be confounded by evolution of phage-resistant variants

• biotyping may be confounded when species are heterogenous for biochemical pathways

molecular typing

based on detection of characteristic nucleotide sequences or proteins

strenths of molecular typing

• culture independent and therefore typically faster

• more sensitive since amplification allows detection of very low numbers of organisms

weaknesses of molecular typing

• antibiotyping remains important and requires culture (presence/absence of an AMR gene is not always confirmation of resistance/sensitivity)

• need to be aware that detection of an organism does not always mean it is responsible for disease

• change in staff training and education

• initial cost of set up i.e equipment, reagants, cryogenic storage etc

identification of a causative agent of an outbreak is confounded by

• presence of other strains in the environment that do not cause disease

• asymptomatic carriage of disease-causing agents by hosts

• need to discriminate between strains genotypically to determine which strains are endemic or epidemic

strains

• subvariant of a species with a common ancestor

• have permanent genetic changes to their DNA content through mutation or HGT (mobile element/recombination)

emergence of a new strain may result in

• commensal becoming pathogen

• pathogen becoming able to colonise and cause disease in a new host

• pathogen become able to cause more sever disease in current host

advantages of PFGE

• good resolution

• whole genome is visualised

• gold standard method widely used

disadvantages of PFGE

• expensive equipment and training required

• long run times

• requires expert interpretation (incomplete digestion, skewed lanes, some species produce nuclease that degrades DNA, other strains modify restriction sites and block digestion)

advantages of ribotyping

• very reproducible

• has been automated

• detects long term evolutionary trends

disadvantages of ribotyping

• less discriminatory than PFGE (fewer bands, not whole genome)

• does not discriminate on short evolutionary timescales such as an outbreak

advantages of RAPD

• Inexpensive, efficient and sensitive — most useful for rapid typing and distinction between unrelated isolates

disadvantages of RAPD

• poor reproducibility

• hard to standardise between labs

• variation in band intensity

advantanges of REP-PCR

• quick and more cost effective than PFGE

disadvantages of REP-PCR

• requires enough Reps in close proximity to generate enough PCR products for discrimination of strains

advantages of AFLP

• no need for known sequences in the genome

• high reproducibility

• many loci are simultaneously analysed

• by changing the selective primers different loci can be analysed

• whole genome analysis is (theoretically) possible

disadvantage of AFLP

• high protocol complexity

advantages of MLVA

• highly reproducible/standardisable

disadvantages of MLVA

• species specific design

advantages of MLST analysis

• cheaper in the early days of sequencing

• data is unambiguous, reproducible between labs, easily transferred and compared between labs, scalable and automated using high throughput sequencing

disadvantages of MLST

• cost is no longer better than whole-genome sequencing

• databases are only available for some pathogens

• significantly impacted by recombination affecting the MLST

advantages of SNP-based phylogeny

• high accuracy and resolution over long timescales for clonal species

• relatively straightforward analysis pipeline

• potentialy impacted by SNPs accrued during lab storage

disadvantages of SNP-based phylogeny

• requires a reference genome sufficiently closely relatd to your set of isolates — especially difficult for novel or non-model organisms

• reference choice can impact the number of SNPs called, limiting reproducibility across studies

• over long timescales removing recombination from panmictic species can essentially result in chucking out the entire dataset

advatanges of K-MER approaches

• by far the highest discriminatory ower f the methods discussed here

• reference-agnostic approach avoids bias

disadvantages of K-MER approaches

• most complicated analysis pipeline requiring technical expertise and computing time

• scientific community still disagrees how many SNPs should be considered a single strain (varies by organism)

phylogeny

• model of the relationships between organisms, genes, proteins, or other structures based on common ancestry

• only makes sense when the characters being compared have a high degree of homology

four common uses of phylogeny

• classification

• grouping of genes, proteins, and other molecular sequences including non-coding sequences.

• epidemiological investigations

• analysis of parallel evolution between host and parasite

redial trees

• all branch lengths indicate sequence diversity

• lenths are proportional to that diversity

dendrogram trees

• only horizontal branches show sequence diversity

• vertival lines show how the horizontal branches are related

• may be shown as rectangular or circular, both are dendrograms

cladogram

• branching tree diagram assumed to be an estimate of a phylogeny where the branches are of equal lengths.

• show common ancestry but do not indicate the amount of evolutionary time seperating taxa.

• usually emphasizes that the diagram represent a hypothesis about the actual evolutionary history of a group

phylogram

• branching tree diagram that is assumed to be an estimate of a phylogeny

• branch lengths are proportional to the amount of inferred evolutionary change based on differences in the sequences being compared

ultrametric tree

• phylogram showing inferred evolutionary change over time

• “molecular clock” is inferred based on the number of sequence changes and the expected rate of change to conver the branch lengths to real-world time

evolutionary time

• the passage of time as measured by the number of mutations introduced into a lineage during replication.

• often assumed to be constant within a phylogeny although we know this is not always the case

root

• most distant branche of a tree

• represent the most recent common ancestor of all taxa at the tips of the tree

outlier

• typically the most divergent sequence from your dataset

• often an ortholog from another closely related species is chosen

midpoint rooting

• find the longest path between two taxa and use the midpoint as the root

• useful when no outgroup is available or when ingroup-outgroup status of taxa is uncertain

clade

• group that includes a common ancestor and all its descendants (living and extinct)

• closely related branches may not represent functionally different taxa: often these are part of a single clone or represent functionally identical proteins

monophyletic

• contains only one continuous evolutionary lineage and no other taxa

• not missing any members of that lineage

polyphyletic

• group with no recent common ancestor

• can think of it as a group where one or more common ancestors are missing

paraphyletic

• containing an ancester and only some of its descendants

• almost like an incomplete monophyletic group

distance matrices

• generate a tree the alignment is converted to a distance matrix comparing each of the sequences in a pairwise fashion

• generated using the probability of a subsitition occuring

  • DNA — equal probability for exchange of nucleotides

  • proteins — probability based on redundancy of genetic code, use ‘point accepted mutation’ matrix for these calculations

disadvantage of neighbour joining method

• conversion to a matrix results in loss of information which is sometimes undesirable. compute time required grows exponentially whick makes this impractical for large datasets

maximum parsimony

• makes use of the optimality criterion that the best tree requires the fewest residue substitiutions to explain the data

advantage of maximum parsimony

• final tree will include all the informative positions in the alignment

disadvantage of maximum parsimony

• several trees may be equally persimonous

maximum likelihood

• most common technique used

• uses probability of substitutions at each position as the optimality criteria

advantage of maximum likelyhood

• final tree will include all the informative positions in the alignment

disadvantage of maximum likelihood

• for large datasets the mostl likely tree will never be found

bayesian methods

• for phylogenies no resolved by maximum likelihood

• involves simulating trees in a markov chain monte carlo simulation and keeping the trees with the hightest probabilities

• posterior probabilities of each branch are written into the final tree

advantages of bayesian methods

• includes all informative positions

disadvantages of bayesian method

• very complex phylogenies may not be fully resolved

bootstrapping

resampling-based method for giving a value to tree branches

• if a branch is highly likely to be real, it will appear many times in the bootstrap analsis and will have a bootstrap value close to 100

• boostrap value close to 0 — most likely to be present by chance alone