Metagenome Flashcards

1
Q

What is genomics?

A

→ The whole cell content

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2
Q

What are early life gut microbiomes linked with?

A

→ Development of allergic conditions such as asthma

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3
Q

What is proteomics?

A

→ Whole cell protein content

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4
Q

What is metabolomics?

A

→ cell metabolite content

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5
Q

How do you purify DNA?

A

→ Culture the organism in isolation

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6
Q

Why in practise is DNA not able to be purified?

A

→ organisms do not live in isolation

→ they are a complex mixture of species

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7
Q

What is a microbiota and what does this include?

A

→ Ecological community of commensal and pathogenic microorganisms
→ bacteria, archae, prostists, fungi and viruses

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8
Q

What is a microbiome?

A

→ Collective genomes of the microorganisms in these communities

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9
Q

What diseases have changes in the microbiome been associated with?

A

→ Cancer
→ Depression

→IBS

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10
Q

What can gut microbiome classify?

A

→ individuals as being obese or lean

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11
Q

What are early life gut microbiomes linked with?

A

→ Development of allergic conditions such as asthma

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12
Q

What are the 4 human microbiomes?

A

→ Gut microbiome
→ Skin microbiome

→ Oral microbiome
→ Vaginal microbiome

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13
Q

What can cure a clostridium difficile infection and why?

A

→ Stool transplant

→ the microbiome in CDI is different to a healthy microbiome

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14
Q

Why is 16S targeted PCR used?

A

→ 16S is a ribosomal component of the 30S subunit in prokaryotes
→ all bacteria have the 16S subunit

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15
Q

Describe how 16S targeted PCR amplification works?

A

→ Sample (urine, blood, etc)
→ DNA is extracted from a mixed population of bacteria in the sample

→ 16S PCR amplification - the 16S gene amplifies
→ sequence the PCR

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16
Q

Why are there biases in 16S PCR?

A

→ Some bacteria purify better than others

→ some sequences amplify better than others

17
Q

What is done with the sequences after 16S PCR?

A

→compared to a database of known 16S sequences

18
Q

What bacteria changes during the first year of life and how?

A

→ Actinobacteria drops after the first years of life

19
Q

What bacteria is present in babies when breastfeeding?

A

→ Bifidobacteria

20
Q

What 2 things do you need to consider when doing PCR on a bacterial variable region?

A

→ If it contains enough information to separate the genus

→ amplicon length

21
Q

Why is sequencing a smaller region better?

A

→ The sequencer can read it twice and any errors will cancel out because the two sequences are combined into one region

22
Q

What are disadvantages to the 16S method?

A

→ It is sensitive to contamination

“Kitome” - reagents can have carry over contaminates

23
Q

What is the 16S method used for?

A

→ Low biomass sample

24
Q

How do you mitigate contamination?

A

→ Randomise the samples
→ Note batch numbers of reagents

→ Sequence negative controls

25
Q

What are the disadvantages of long reads?

A

→ they have higher error rates and introduce noise

26
Q

What is whole genome shotgun sequencing?

A

→ Sequencing all the genes not just the 16S

27
Q

How does whole genome shotgun sequencing work?

A

→the entire genome is broken into small fragments of DNA
→short fragments are sequenced and assembled together by computer that find overlaps

→ you can assess the DNA taxonomically
→ overlay it on pathways
→ changes in metabolic pathways between species

28
Q

What are you looking for in whole genome shotgun sequencing?

A

→ changes in metabolic pathways between species

29
Q

What are the 3 issues with whole genome shotgun sequencing?

A

→ Host cells are in excess
→ no amplification step to enrich for bacterial DNA

→ sample dependent

30
Q

How do you enrich without amplification (pre extraction)?

A

→ Differential lysis of mammalian cells - bacteria have different cell walls
→ put reagents to lyse the mammalian cells but not bacteria

31
Q

Why can there be a bias with lysing mammalian cells?

A

→ Bias towards gram +ve bacteria which have a thicker cell wall

32
Q

How do you enrich without amplificaiton (post extraction)?

A

→ Enzymatic degradation of methylated nucleotides which target mammalian DNA
→ bias against AT rich bacterial genomes

33
Q

How is metagenomics used in diagnostic microbiology?

A

→culture isolate and then identify using matrix assisted laser desorption/ionization (MALDI)
→Can identify hard to culture organisms in patient samples

34
Q

How can metagenomics be used in public health?

A

→Infection control and outbreak management

→Surveillance of antimicrobial resistance in the food supply

35
Q

How do you decide which variable to choose?

A

Phylogenetic signal- (shorter sample will correct mistakes as read twice/overlap).

Amplicon length- changes with intro of new sequencing platforms

36
Q

What are the two ways of assessing shotgun sequences?

A
  1. Assembly

2. Binning

37
Q

What is binning?

A

process of grouping reads orcontigsand assigning them to individualgenome by comparing to databases