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Genomics > The Metagenome > Flashcards

The Metagenome Flashcards

1
Q

What is metagenomics?

A

This is the study of genetic material recovered directly from environmental or biological systems/compartments

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2
Q

What is the significance of metagenomics?

A
  • Unbiased view of taxanomic diversity in a sample
  • Not limited by ability to culture
  • Overall view of gene content in a sample
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3
Q

How many base pairs are sequenced using metagenomics?

A

1.045 billion base pairs sequenced

elucidate the gene content, diversity, and relative abundance of the organisms

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4
Q

What are some of the significant findings of metagenomics

A

estimated to derive from at least 1800 genomic species
identified 148 previously unknown bacterial phylotypes
identified over 1.2 million previously unknown genes

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5
Q

What is microbiota?

A

ecological community of commensal and pathogenic microorganisms
Includes bacteria archaea, protists, fungi and viruses

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6
Q

What is the microbiome?

A

The collective genomes of microorganisms in these communities

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7
Q

Name some environemntal microbiota

A

Deep sea microbiome
Soil microbiome
Hospital microbiome
Subway microbiome

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8
Q

What are some examples of human microbiota?

A

Gut microbiome
Skin microbiome
Oral microbiome
Vaginal microbiome

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9
Q

How diverse are the human microbiota?

A

Taxonomic diversity varies by body site

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10
Q

How does microbiome differ from person to person?

A

Microbiome unique to each individual, even between twins

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11
Q

What does a change in microbiome suggest?

A

Changes in the microbiome have been associated with multiple human illnesses, e.g. Irritable Bowel Syndrome, depression, cancer

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12
Q

What is the significance of gut microbiome?

A

Gut microbiome can classify individuals as lean or obese with >90% accuracy
Early-life gut microbiomes linked to development of allergic conditions e.g. asthma

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13
Q

What is CDI?

A

Clostridium difficile infection (CDI) is a disease of the large intestine caused by toxins produced by the spore forming bacterium Clostridium difficile

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14
Q

How does stool microbiota differ in CDI?

A

Stool microbiome during Clostridium difficile infection (CDI), quite different from healthy stool microbiome
CDI has greater effect on stool microbiome than host genetic factors

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15
Q

How CDI cured?

A

Faecal microbiota transplant is able to cure CDI

Restoration of the stool microbiome to that of healthy state is rapid following transplantation

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16
Q

What is the relevant factor of microbiota in causing disease?

A

The relative abundance of organisms may not be as important gene content

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17
Q

What are the technological approaches to metagenomics?

A
  • Targeted PCR amplification

- Whole Genome Shotgun Sequencing

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18
Q

Describe the PCR technique used in metagenomics

A

16S rRNA, bacteria

Internal transcribed spacer (ITS), 18S rRNA, eukaryotes

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19
Q

Outline the benefits of Whole Genome shotgun sequencing in metagenomics

A

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

20
Q

What is the 16s ribosomal RNA?

A

16S ribosomal RNA is component of 30S small subunit of prokaryotic ribosome

21
Q

Outline the 16s Targeted PCR amplification workflow

A
  1. Sample selection
  2. DNA extraction
  3. 16s PCR amplification
  4. Sequencing
  5. Analysis
22
Q

Name some of the 16s databases

A

Greengenes
Silva
RDP

23
Q

Give examples of open source analysis pipelines available

A

QIIME
Mothur
DADA2

24
Q

What are the 4 main metagenomic technologies available

A

Roche454
Illumina HiSeq
IonTorrent PGM
Illumina MiSeq

25
Q

Which regions are commonly used in 16s Targeted PCR amplification?

A

Different studies differ
V1 - V2 and V1 - V3 are very common
but there is no consensus for which region to use

26
Q

How does gut microbiota change with age?

A

<4 yrs old have lots of actinobacteria (generally bifidobacterium well known baby gut bacteria)
As you get older bifidobacterium goes down, bacteroides comes up

27
Q

What are the 2 factors taken into consideration when choosing which variable region to sequence?

A
  • Phylogenetic signal

- amplicon length

28
Q

Describe how phylogenetic signals in bacteria determine which variable region to choose?

A

Similarities cluster together, differences cluster further apart
e.g.
V1-V2 reds (Staphylococcus epidermidis) and blue (staphylococcus aureus) are separated quite clearly
V4 tree: reds and blues are very mixed

to study skin biome use V1-V2

29
Q

How does amplicon length affect the variable region chosen?

A

Errors present in sequences (by random). The error in the middle will be corrected due to overlap
Errors not overlapped will remain in sample ∴ data won’t be as good
where there is 100% overlap the whole sequence is corrected ∴ provides a much more accurate estimation of genes present

30
Q

Where inthe genome 16s rRNA found?

A

16S rRNA gene found in all bacteria

31
Q

How susceptible to contamination is the 16s rRNA gene?

A

Method very sensitive to contamination from
- Environment
- Operator
- Reagents
Especially important for low biomass samples

32
Q

Why are low biomass samples more susceptible to contamination?

A

E.g. fecal sample has lots of bacteria material ∴ contamination will be at a smaller proportion - less effect
Skin swabs have a small bacteria presence so contamination will have a greater effect on results

33
Q

How can we mitigate potential contamination?

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

Why s choosing the correct variable region important?

A

Choice of variable region determines resolution

Less reliable below genus level

35
Q

What are the new full length 16s technologies available?

A

New long read technology enable full length 16S sequencing
PacBio
Nanopore

36
Q

What is the drawback of full length 16s sequencing?

A

However, higher error rates of long read technologies introduce noise
Development ongoing

37
Q

How does WGS differ from 16s sequencing?

A

In 16s rRNA amplification only sequence one gene

WGS sequences across all genomes at the same time - don’t always align but are made up of different bacterial species

38
Q

How does WGS sequencing allow us to see genes?

A

WGS Shotgun is assembled

After assembly we can form our phylogenetic tree to find relative abundances of bacteria and genes present.

39
Q

What other analysis does WGS allow?

A

Gene prediction can also occur as we have sequenced all genomes - can look at pathways and which genes contribute to which pathway in certain individuals genes in pathways may be accelerated etc.

40
Q

Why is WGS still not 100% reliable?

A

Host cells often in excess in the sample as amplification not done
No amplification step to enrich for bacterial DNA
need to ensure we’re not just sequencing host cell

41
Q

Which samples are more likely to be contaminated by hosts using WGS?

A

Faecal: <10% human reads

Saliva, nasal, skin samples: >90% human reads

42
Q

What steps are taken to enrich the sample without amplification in WGS?

A

Pre-extraction

Post-extraction

43
Q

Describe pre-extraction?

A

Differential lysis of mammalian cells
Enrichs for intact microbial cells
Potential bias towards gram-positive bacteria

44
Q

What is post-extraction?

A

Enzymatic degradation of methylated nucleotides targets mammalian DNA
Bias against AT rich bacterial genomes

45
Q

What open source analysis pipelines are available for WGS?

A

MG-RAST
Metaphlan
Megan

Genomics (20 decks)

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