Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

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

Lecture 12: Principles of genome and PCR based diagnostics Flashcards

You may prefer our related Brainscape-certified flashcards:
Biology 101 flashcards
1
Q

What does the Sanger sequencing reaction require?

A

Template DNA to sequence

Primer

DNA polymerase

Nucleotides

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What is the primer in the Sanger sequence for?

A

Short DNA sequence complementary to template DNA

Anneals to a template

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

How does the Sanger sequencing work?

A

Incorporation of ddNTPs into the new strands

Causes chain termination

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What is Sanger sequencing also called?

A

Chain termination or dideoxy method

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What is the ratio of ddNTP to dNTP?

A

1 dNTP: 100 ddNTP

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is the run time of Sanger sequencing?

A

1-3 hours

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What is the average read length of Sanger Sequencing?

A

700 base pairs

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

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

A

Sanger sequencing

99.99%

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What is the high-throughput sequencing the pinnacle of?

A

Strain characterization

Epidemiological analysis

Conventional typing approaches

Resistance gene detection

Identification

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

How is the base chemically modified in Illumina sequencing?

A

Remove the fluorophore

Allow another fluorescent base to be added

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What is base call accuracy?

A

The probability of a correct base call

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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%

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

What are contigs?

A

Putting together overlapping reads to build longer sequences

Known as Contigs

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

What are the long read sequencers?

A

PacBio

Oxford Nanopore

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

What is the process of Oxford nanopore technology?

A
  1. Construct library
  2. Sequence library
  3. Analyse data
  • NO AMPLIFICATION
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

What is the average read length of Oxford Nanopore technology?

A

Very long reads

Up to 4 MB

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

What is the accuracy of Oxford Nanopore technology?

A

95-99.9%

26
Q

Why is typing needed?

A

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
Q

What are the advantage of studying genomics?

A

Can query the whole genome or just a gene

Precision
Sensitivity
Speed
Personalisation

28
Q

Why should genomic data be used in an outbreak?

A

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
Q

What is the definition of molecular and genomic epidemiology?

A

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
Q

What is a single nucleotide polymorphism (SNP)?

A

A genomic variant at a single base position in the DNA

31
Q

What are principles of genomic epidemiology?

A

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
Q

What does genomic epidemiology sequencing allow for?

A

Generate genomic fingerprint reflective of the organism

Detect all accumulating mutations in genome

Compare genomic fingerprints

33
Q

How does genomic epidemiology work?

A

Using sequencing data, determine and visualize relationships

SNPs provide basic unit of difference

Tree is indicative of relatedness of different isolates

34
Q

What is SNP calling?

A

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
Q

Why is a choice of reference crucial in SNP calling?

A

High-quality, closed reference genome

Ideally closely related to set of traits under investigation

36
Q

What are the key strength and weakness of conventional MLST?

A

Low sensitivity - good for phylogenetic analysis

High robustness - not good enough for outbreak investigation

37
Q

What is the definition of the core genome?

A

Set of genes that is found in every member of biological species

38
Q

How does Core genome MLST work?

A

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
Q

What is the difference between core genome MLST and whole-genome MLST?

(cgMLST vs wgMLST)

A

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
Q

What is an accessory genome?

A

Gene set shared within only one or some strains

41
Q

What is the difference between SNP and cgMLST?

A

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
Q

What 6 categories can virulence factors be differentiated into?

A

Adherence and colonization factors

Type I and VI secretion systems

Immune evasion factors

Toxins

Siderophores for iron absorption and invasion genes

43
Q

Why is whole genome sequencing detecting virulence factors important?

A

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
Q

What is the mobilome?

A

Set of mobile genetic elements that strongly influence bacterial phenotypes by their presence of absence

45
Q

What are the difference types of mobile genetic elements and what do they force?

A

MGEs force exchange of genetic material between different bacteria

Different types: insertion sequences, bacteriophage, integrative and conjugative elements, plasmids

46
Q

Why can mobile genetic elements of unrelated bacteria be easily detected?

A

They often exhibit G +C contents that differ to some extents from that of their hosts

Indicating earlier events of lateral gene transfer

47
Q

How can antimicrobial resistance be identified in whole-genome sequencing data?

A

Using assembly-based approaches

Using BLASTIN-based tools comparing derived draft genome to AMR reference gene databases

48
Q

Why are antimicrobial resistance databases heavily biased towards easy-to-cultivate pathogens?

A

By using current antimicrobial resistance databases

Novel or remote homologous AMR genes from less well studied bacteria might be missed

49
Q

Why is complete replacement of phenotypic AMR measurement by molecular AMR prediction not advised?

A

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
Q

Why does phenotypic testing of a set of representative diverse strains need to be maintained in antimicrobial resistance testing?

A

Ensures genotypic AMR results do not diverge from the true AMR phenotype over time

51
Q

What is a overview of whole-genome sequencing?

A

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
Q

What are the key points from the whole genome study on Acinetobacter baumannii?

A

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
Q

What can be done for organisms that are difficult to grow or unable to grow in culture?

A

Targeted sequencing

Shotgun sequencing

54
Q

What is targeted sequencing?

A

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
Q

What is shotgun sequencing?

A

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
Q

What is a limitation of shotgun metagenomics?

A

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
Q

How has shotgun metagenomics been applied to the management and treatment of C. Difficle?

A

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
Q

What is the future of genomics in a clinical setting?

A

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
Q

What do current shotgun sequencing bioformatics do and need improving on?

A

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
Q
A

LF255 - Clinical Microbiology (10 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions