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LF255 - Clinical Microbiology > Lecture 12: Principles of genome and PCR based diagnostics > Flashcards

Lecture 12: Principles of genome and PCR based diagnostics Flashcards

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1
Q

What does the Sanger sequencing reaction require?

A

Template DNA to sequence

Primer

DNA polymerase

Nucleotides

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2
Q

What is the primer in the Sanger sequence for?

A

Short DNA sequence complementary to template DNA

Anneals to a template

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3
Q

How does the Sanger sequencing work?

A

Incorporation of ddNTPs into the new strands

Causes chain termination

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4
Q

What is Sanger sequencing also called?

A

Chain termination or dideoxy method

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5
Q

What is the difference between ddNTP and dNTP?

A

ddNTPs are missing a hydroxyl group on the 3’ carbon

Cannot form a bond with a 5’ phosphate

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6
Q

What is the ratio of ddNTP to dNTP?

A

1 dNTP: 100 ddNTP

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7
Q

What is the process of Sanger sequencing?

A

ddNTPs fluorescent in 4 different colours

Single reaction gives information on each base

Reaction are run on capillary gels - read by lasers and detectors as sample passes detection window

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8
Q

What is the run time of Sanger sequencing?

A

1-3 hours

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9
Q

What is the average read length of Sanger Sequencing?

A

700 base pairs

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10
Q

What sequencing method is considered the ‘gold standard’ and what is the accuracy?

A

Sanger sequencing

99.99%

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11
Q

What is the 2024 Sequencing Landscape?

A

Thermofisher (short read sequences, less than 1000 bases)

Illumina

PacBio

Oxford Nanopore (long read sequencers, greater than 1000 bases)

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12
Q

What is the high-throughput sequencing the pinnacle of?

A

Strain characterization

Epidemiological analysis

Conventional typing approaches

Resistance gene detection

Identification

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13
Q

What is Illumina sequencing?

A

Sequencing by synthesis method

Using primer annealing to the DNA template that is extended by polymerase

Fluorescently labelled nucleotides

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14
Q

How is the base chemically modified in Illumina sequencing?

A

Remove the fluorophore

Allow another fluorescent base to be added

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15
Q

What is the process of Illumina sequencing?

A
  1. Library preparation (adapters)
    *added sequences hybridise to homologous primers on surface of sequencing flow cell
  2. PCR: bridge amplification (enhances signal)
    *making copies of all genes
  3. Sequencing by synthesis (copy template strands)
    *make DNA bind and bind to hybridised primers
  4. Signal detection (1 image per cycle)
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16
Q

What is base call accuracy?

A

The probability of a correct base call

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17
Q

What is the run time, average read length and accuracy of Illumina sequencing?

A

Run time: 1-3 days

Read length: 2x75 - 2x300 bases

Accuracy: 99.9%

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18
Q

What is the disadvantage of short read sequencing?

A

Needs to be fragmented for sequencing

Million of 150 to 300 nucleotide long fragments

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19
Q

What are contigs?

A

Putting together overlapping reads to build longer sequences

Known as Contigs

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20
Q

What is genome assembly?

A

Search for sequence homologies among reads

Put together overlapping reads

Build longer sequences

Compares contigs to closely related genome to determine order and produce scaffold

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21
Q

What are the long read sequencers?

A

PacBio

Oxford Nanopore

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22
Q

What is the process of Oxford nanopore technology?

A
  1. Construct library
  2. Sequence library
  3. Analyse data
  • NO AMPLIFICATION
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23
Q

How does Oxford Nanopore technology work?

A

Naturally occurring membrane proteins that form pores are modified and incorporated into artificial membranes

Added ionic buffer

Measure current passing though membrane, anything passing through pore changes current

Sensors for each pore can measure change - passing through current

Signal decoded using base-calling algorithms to determine DNA or RNA sequence in real time

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24
Q

What is the average read length of Oxford Nanopore technology?

A

Very long reads

Up to 4 MB

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25
What is the accuracy of Oxford Nanopore technology?
95-99.9%
26
Why is typing needed?
Greatly enhance insights into microbial population and epidemiology and microbial diversity Widely used in diagnostics, genomics, and pathogenesis related with microbiology research Gives ability to accurately distinguish among strains of infection pathogens
27
What are the advantage of studying genomics?
Can query the whole genome or just a gene Precision Sensitivity Speed Personalisation
28
Why should genomic data be used in an outbreak?
Monitor trends at national level - Emergence of new infectious agents - Predicting severity and size - Trends after interventions - Understand historical trends Better understand epidemiology at local level - Identify source of transmission - Investigate clusters of transmission - Provide evidence for and against suspected transmission - Reveal important, unsuspected clusters
29
What is the definition of molecular and genomic epidemiology?
Use of pathogen molecular and genomic data to determine the distribution and spread of an infectious disease in a specified population and application of this information to control health problems Molecular data information on specific genes Genomic data information on whole genome
30
What is a single nucleotide polymorphism (SNP)?
A genomic variant at a single base position in the DNA
31
What are principles of genomic epidemiology?
Replication and repair processes (stochastic errors, DNA polymerase proofreading, DNA damage response) Genomic factors (transcription, chromatin state, replication time) Environmental exposures (UV light, chemotherapy agents)
32
What does genomic epidemiology sequencing allow for?
Generate genomic fingerprint reflective of the organism Detect all accumulating mutations in genome Compare genomic fingerprints
33
How does genomic epidemiology work?
Using sequencing data, determine and visualize relationships SNPs provide basic unit of difference Tree is indicative of relatedness of different isolates
34
What is SNP calling?
Identification of SNPs that vary among strains Detected by mapping sequence reads against reference genome Relatedness based on number of SNP differences between isolates
35
Why is a choice of reference crucial in SNP calling?
High-quality, closed reference genome Ideally closely related to set of traits under investigation
36
What are the key strength and weakness of conventional MLST?
Low sensitivity - good for phylogenetic analysis High robustness - not good enough for outbreak investigation
37
What is the definition of the core genome?
Set of genes that is found in every member of biological species
38
How does Core genome MLST work?
Genome data is aligned to a scheme Comparison will yield either allele number of an allele sequence already present in a scheme Allele profile produced Total number of differently assigned allele numbers between a pair of samples determines relatedness with each other
39
What is the difference between core genome MLST and whole-genome MLST? (cgMLST vs wgMLST)
wgMLST extension to cgMLST cgMLST uses core genome loci and accessory loci wgMLST provides higher resolution as large set of loci used wgMLST more useful when comparing closely related isolates
40
What is an accessory genome?
Gene set shared within only one or some strains
41
What is the difference between SNP and cgMLST?
SNP and MLST analyses are congruent Both approaches are well suited and commonly applied for outbreak analyses SNP has higher discriminatory power - cgMLST reports allele difference between 2 strains, doesn't provide information on number of mutations SNP calling best for analysis of detailed evolutionary models
42
What 6 categories can virulence factors be differentiated into?
Adherence and colonization factors Type I and VI secretion systems Immune evasion factors Toxins Siderophores for iron absorption and invasion genes
43
Why is whole genome sequencing detecting virulence factors important?
Makes it possible to identify new gees or gene variants that confer virulence to bacteria It is a relevant indication for the pathogenicity of most bacteria
44
What is the mobilome?
Set of mobile genetic elements that strongly influence bacterial phenotypes by their presence of absence
45
What are the difference types of mobile genetic elements and what do they force?
MGEs force exchange of genetic material between different bacteria Different types: insertion sequences, bacteriophage, integrative and conjugative elements, plasmids
46
Why can mobile genetic elements of unrelated bacteria be easily detected?
They often exhibit G +C contents that differ to some extents from that of their hosts Indicating earlier events of lateral gene transfer
47
How can antimicrobial resistance be identified in whole-genome sequencing data?
Using assembly-based approaches Using BLASTIN-based tools comparing derived draft genome to AMR reference gene databases
48
Why are antimicrobial resistance databases heavily biased towards easy-to-cultivate pathogens?
By using current antimicrobial resistance databases Novel or remote homologous AMR genes from less well studied bacteria might be missed
49
Why is complete replacement of phenotypic AMR measurement by molecular AMR prediction not advised?
Bacterial strains continue to evolve and new resistance mechanisms emerge that are not represented in AMR databases or in the datasets used to train machine learning models
50
Why does phenotypic testing of a set of representative diverse strains need to be maintained in antimicrobial resistance testing?
Ensures genotypic AMR results do not diverge from the true AMR phenotype over time
51
What is a overview of whole-genome sequencing?
The process of sequencing and assembling the microbial genome of an organism of interest Requires culture and isolation of the organism Limitation for organisms that are difficult to grow or unable to grow in culture
52
What are the key points from the whole genome study on Acinetobacter baumannii?
Nosocomial outbreak of Acinetobacter baumannii Typed by PHE, designated unique pulsotype 27 Defined 7 major outbreak genotypes Able to follow genotypes of spreading between wards consistently with timeline of patients in wards
53
What can be done for organisms that are difficult to grow or unable to grow in culture?
Targeted sequencing Shotgun sequencing
54
What is targeted sequencing?
Selection or enrichment process performed for an organism or a group of organisms of interest either prior to or after the library preparation process Selection via PCR or probe hybridisation Minimises human DNA interference and provides higher sensitivity of detection in sample types
55
What is shotgun sequencing?
Sequencing of all nucleic acid in a sample to identify ALL bacteria, fungi, viruses, and parasites in a test Used to detect infection from specimen types that are normally sterile such as CSF, blood and joint fluid Can detect infectious agents in specimen types with a documented microbiome Limitation: background noise or interference of human nucleic acid or the resident microbiome
56
What is a limitation of shotgun metagenomics?
Only small proportion of reads are non-human Only subset may correspond to potential pathogens Sensitivity is critically dependent on the level of background and distinguishing from normal microbiome
57
How has shotgun metagenomics been applied to the management and treatment of C. Difficle?
Studies characterised microbiomes of patients and lead to the development of bacterial probiotic mixtures that can be administered as pills These can be administered as pills for prophylaxis or treatment of C. Diff associated disease
58
What is the future of genomics in a clinical setting?
Only for select few clinical microbiology laboratories with budget and personnel to make it possible Depended on price to make it more readily available to regional labs Turn around time beating culture-dependent methods Improving samples processing to overcome issues with contaminating human DNA and low input inherent of clinical samples Need improvements in understanding of genotype
59
What do current shotgun sequencing bioformatics do and need improving on?
Detect multiple pathogens that are present within a given sample Find it difficult to assemble the full genome of a pathogen Improvements leads to confident pathogen assembly of a complete genome will give ability to predict virulence and antimicrobial resistance markers
60

LF255 - Clinical Microbiology (10 decks)

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