7 WGS Flashcards by Yasmin Yau

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)

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what are the four main methods used in diagnostic microbiology

microscopy
culture
serology
molecular

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how does most treatment start

empirically

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what are the problems with methods in diagnostic microbiology

time consuming
expensive
labour-intensive
organism or sample-type specific
poor discrimination

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why can diganostic microbiology be time-consuming

bacteria need time to grow

unusual organism – sent to reference lab

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Genome

complete set of genes or genetic material present in cell or organism (DNA/RNA)
blueprint describing the characteristics of an organism

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what does the genome information provide

record of ancestry revealing genetic relationships between members of the same species or more distant ones

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Central dogma

information flow – DNA genes transcribed to mRNA then translated into protein

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what are the genes like in bacterial genome

> Short intergenic regions
Little repeat / non-coding DNA
Introns are v. rare

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how are genes organised in bacterial genome

operons

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Bacterial genome organisation - Chromosome

Typically - a single circular chromosome (always DNA) which is negatively supercoiled in order to pack it in

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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

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linear chromosome examples

Streptomyces

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two unique circular chromosome example

Vibrio cholerae

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multiple and a mix of circular and linear chromosome example

Agrobacterium tumefaciens

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Plasmid replication

Independent autonomously replicating DNA molecule

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what do plasmids encode for

‘non-essential’ genes, but relate to virulence (e.g. tetanus toxoid encoded by plasmid) or adaptive advantage

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sequencing method example

sanger sequencing

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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

1. What organism is causing the infection? 2. What drugs can be used to treat it? 3. 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