7 WGS Flashcards
what does the current paradigm used in diagnostic microbiology consist of
- detection (or not) of the presence of a pathogen
- identification
- antibiotic susceptibility testing (and surveillance)
- epidemiological typing (and surveillance)
what are the four main methods used in diagnostic microbiology
- microscopy
- culture
- serology
- molecular
how does most treatment start
empirically
what are the problems with methods in diagnostic microbiology
- time consuming
- expensive
- labour-intensive
- organism or sample-type specific
- poor discrimination
why can diganostic microbiology be time-consuming
bacteria need time to grow
unusual organism – sent to reference lab
Genome
complete set of genes or genetic material present in cell or organism (DNA/RNA)
blueprint describing the characteristics of an organism
what does the genome information provide
record of ancestry revealing genetic relationships between members of the same species or more distant ones
Central dogma
information flow – DNA genes transcribed to mRNA then translated into protein
what are the genes like in bacterial genome
> Short intergenic regions
Little repeat / non-coding DNA
Introns are v. rare
how are genes organised in bacterial genome
operons
Bacterial genome organisation - Chromosome
Typically - a single circular chromosome (always DNA) which is negatively supercoiled in order to pack it in
what can chromosome of bacterial genome be like
Occasionally linear
Occasionally two unique circular
Can even be multiple and a mix of circular and linear
linear chromosome examples
Streptomyces
two unique circular chromosome example
Vibrio cholerae
multiple and a mix of circular and linear chromosome example
Agrobacterium tumefaciens
Plasmid replication
Independent autonomously replicating DNA molecule
what do plasmids encode for
‘non-essential’ genes, but relate to virulence (e.g. tetanus toxoid encoded by plasmid) or adaptive advantage
sequencing method example
sanger sequencing
sanger invention
Sanger came up with chain terminator – so DNA polymerase cannot add in next nucleotide – irreversibly stops DNA replication
what is used in the sanger reaction
Labelled primer annealed to template DNA
Primer (radio labelled) extended by DNA polymerase until a ddNTP is incorporated terminating chain extension
Fragments separated by size on an electrophoresis gel and image collected via labelled primers
1 bp resolution
Sanger sequencing improvements
Automated Sanger sequencing:
Fluorescent labels to replace radio-isotopes
Capillary gel electrophoresis
Sequencing methods: Whole-Genome Shotgun Sanger Sequencing
Take the purified DNA
Random shearing
Selected certain size fragment
Clone into plasmid and transform into E. coli cells
Each E. coli has a different potion of the bacteria plasmid
Need thousands of colonies to cover all the bacterial plasmid
Plasmid prep
Sequence ‘insert’ in each clone using primers on either side
QC, assembly and annotation
Sequence assembly
Assembly = reads are merged into a longer DNA sequence in the attempt to reconstruct the original DNA sequence
what does sequencing generate
millions of reads
Whole-Genome Shotgun Sanger Sequencing – Problems!!
- Onerous colony picking and plasmid preparation
- Expensive
- Amplified templates generated in vivo, so genes toxic to E. coli not represented in sequence libraries
Sequencing methods: High-throughput sequencing
platforms allow many millions of target DNA molecules to be sequenced simultaneously, resulting in substantial reductions in cost and in the time taken to produce a whole genome sequence
what is amplified in high-throughput screening
amplification of individual clonal templates
based on chemistry not biology – DNA polymerase does the ‘work’ rather than E. coli
High Throughput Sequencing – Problems!!
- smaller read length
- computational costs
- cannot achieve accuracy in long repeat assembly
- difficult to obtain finished genome
Nanopore sequencing
One forms pore in membrane other is a DNA unwinding enzyme
Add DNA in and starts passing it through pore
As each base pair goes through form current
what are the nanopores
MinION
GridION
read-length of GridION
No limit to read-length, but typically 100s kb
Single molecule sequencing - Problems!!
- Higher error rate, but makes assembly much easier
how are bacterial genomes generated
using a mix of ‘high- throughput’ sequencing (for accuracy) and ‘single molecule’ re- sequencing (to help assembly), but as accuracy improves single molecule sequencing will become the norm
what is whole genome sequencing
the process of determining the complete sequence of an organism’s genome
What clinicians need to know asap
- What organism is causing the infection?
- What drugs can be used to treat it?
- How is it related to other similar infections?
what does WGS provide in detecting organism causing infection
WGS can provide (high resolution) identification of bacterial pathogen
why is WGS not used to identify organisms
Still need to grow and isolate bacteria (24 h or longer)
what drugs can be used to treat - WGD and detecting resistance
WGS can detect the presence of resistance genes & mutations
what is necessary if using WGS for resistance detection
you need to know what you are looking for
why is WGS not used for resistance detection
- genetic basis of many resistance mechanisms is not known
- Need comprehensive and validated databases of clinically relevant genotype- phenotype correlations – not available yet
what can WGS provide in infection detection
WGS can provide (v. high resolution) typing of a bacterial pathogen
cons of determining how related it is to other similar infections
Unlikely to improve outcomes for those patients in real-time
Whole genome sequencing: advantages over current methods
- can be used for all bacteria due to ‘generic’ genome nature
- better discriminate between pathogens, enable outbreaks to be resolved/ruled out quicker
- provide description of wide range of clinically + epidemiological characteristics in one test
Whole genome sequencing: potential problems
cost
time
data analysis - converting massive amount WGS yield
benefits of WGS - working out how similar to related infection
- determine the likely sources + transmission paths
- identify DNA sequences unique to outbreak strain - can be used to develop more rapid molecular assay
