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Genomics > The Metagenome > Flashcards

The Metagenome Flashcards

1
Q

Genomics

A

Whole cell gene content

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2
Q

Transcriptomics

A

Whole cell gene expression

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3
Q

Proteomics

A

Whole cell protein content

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4
Q

Metabolomics

A

Whole cell metabolite content

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5
Q

Why is genomics sometimes a difficult concept?

A

Concept that organisms do not live in isolation and there is a complex interaction between environments and species

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6
Q

Metagenomics

A

The study of genetic material recovered directly from environmental or biological systems/compartments.

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7
Q

Microbiota

A

Microbiological content of a sample for example the ecological community of commensal and pathogenic microorganisms including bacteria, archaea, protists, fungi and viruses

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8
Q

Microbiome

A

Genomic content of a sample - the collective genomes of the micro-organisms in these communities

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9
Q

Examples of environmental microbiomes

A
  • Deep sea microbiome
  • Soil microbiome
  • Hospital microbiome
  • Subway microbiome
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10
Q

Examples of human microbiomes

A
  • Gut microbiome
  • Skin microbiome
  • Oral microbiome
  • Vaginal microbiome
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11
Q

What varies by body site? And what does this mean?

A

Taxanomic diversity - Ths refers to the type of bacteria depends on where in the body location is being analysed

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12
Q

Most common bacteria in the stomach

A

H. pylori

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13
Q

What has a change in the microbiome been associated with?

A

Associated with multiple human illnesses, e.g. irritable bowel syndrome, depression, cancer

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14
Q

What does analysing the gut microbiome in individuals help to do?

A

To classify individuals as lean or obese with approx. 90% accuracy

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15
Q

In early life, what is the gut microbiome thought to be linked to?

A

The development of allergic conditoins e.g. asthma

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16
Q

How is stool microbiome during clostridium difficile infection (CDI) different to healthy CDI?

A

CDI has a greater effect on stool microbiome than host genetic factors.

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17
Q

What is able to cure CDI?

A

Faecal microbiota transplant to restore healthy bowels. It returns to the healthy state rapidly following transplantation.

18
Q

What are the two technological approaches to metagenomics?

A
  1. Targeted PCR amplification

2. Whole genome shotgun sequencing

19
Q

What is targeted PCR amplification?

A

16s rRNA, bacteria done more in bacteria than eukaryotes

Internal transcribed spacer (ITS), 18S rRNA eukaryotes

20
Q

What is 16S ribosomal RNA?

A

It is a component of 30S small subunit of prokaryotic ribosome

21
Q

What is the structure of 16S rRNA?

A

1542 base pairs with 9 variable regions and alternating conserved regions

22
Q

What do variable regions determine?

A

They determine the phylogenetics (which bacteria is present)

23
Q

Summarise the workflow of 16S targeted PCR amplification

A 
Sample collection
DNA extraction 
16S PCR amplification 
Sequencing 
Analysis
24
Q

What can sequences in the 16S rRNA determine?

A

It can determine how many bacterial species are in the sample

25
Q

Describe briefly the changes in gut microbiota by age

A

Actinobacteria - is the highest when you are youngest and decreases with age
Bacteriodietes - highest when you’re youngest and decreases with age
Firmcutes - increases with age remains fairly constant until later life where starts to decrease
Proteobacteria mostly unchanged throughout life may decrease in old age

26
Q

Which variable region is commonly used for sequencing?

A

V1 - V2 and V3 - V4

27
Q

What factors are considered when choosing a variable region?

A
  • Phylogenetic signal

- Amplicon length

28
Q

What is the phylogenetic signal?

A

The genetic tree depending on the area being studied.

29
Q

What is the amplicon length?

A

Areas that overlap being corrected. If they do not overlap, then it won’t be corrected. There’s a lot of noise.

30
Q

What is long read technology?

A

Tech that can read more kB. It will be able to read V1 - V9.

31
Q

What is 16S targeted PCR amplification sensitive to?

A

Contaimination from the environment, operator or reagents.

32
Q

Why is understanding the contamination of 16S targeted amplification important?

A

It is important for low biomass samples.
For example, in faecal samples, there is a lot of bacteria whereas in skin samples there is less so it is more damaging if contaminated.

33
Q

How can potential contamination be mitigated?

A
  • Randomise samples
  • Note batch numbers of reagents
  • Sequence negative controls
34
Q

What does the choice of variable region determine?

A

The resolution

35
Q

Why is there a higher error rate with long read technologies?

A

Introduce noise

36
Q

Describe the whole genome shotgun workflow

A

DNA can either go through 16S rRNA amplicon or WGS shotgun
Through WGS it forms an assembly. This assembly can be used to create a taxanomic diversity or for gene prediction and other pathways.

37
Q

How is samples enriched without amplification pre-extraction?

A
  • Differential lysis of mammalian cells
  • Enriches for intact microbial cells
  • Potential bias towards gram-positive bacteria
38
Q

How are samples enriched without amplifcation post extraction?

A
  • Enzymatic degradation of methylated nucleotides targets mammalian DNA
  • Bias against AT rich bacterial genomes
39
Q

Benefits and Cons of Targeted 16S PCR amplification

A

Assess taxanomic diversity in sample

Biased, only bacteria

40
Q

Benefits of WGS sequencing

A

Assess taxanomic diversity in sample
Assess composite gene functions in sample
Unbiased, all micro-organisms

Genomics (19 decks)

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