The Metagenome

Question 1

Genomics

Answer 1

Whole cell gene content

Question 2

Transcriptomics

Answer 2

Whole cell gene expression

Question 3

Proteomics

Answer 3

Whole cell protein content

Question 4

Metabolomics

Answer 4

Whole cell metabolite content

Question 5

Why is genomics sometimes a difficult concept?

Answer 5

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

Question 6

Metagenomics

Answer 6

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

Question 7

Microbiota

Answer 7

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

Question 8

Microbiome

Answer 8

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

Question 9

Examples of environmental microbiomes

Answer 9

• Deep sea microbiome
• Soil microbiome
• Hospital microbiome
• Subway microbiome

Question 10

Examples of human microbiomes

Answer 10

• Gut microbiome
• Skin microbiome
• Oral microbiome
• Vaginal microbiome

Question 11

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

Answer 11

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

Question 12

Most common bacteria in the stomach

Answer 12

H. pylori

Question 13

What has a change in the microbiome been associated with?

Answer 13

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

Question 14

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

Answer 14

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

Question 15

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

Answer 15

The development of allergic conditoins e.g. asthma

Question 16

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

Answer 16

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

Question 17

What is able to cure CDI?

Answer 17

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

Question 18

What are the two technological approaches to metagenomics?

Answer 18

1. Targeted PCR amplification

2. Whole genome shotgun sequencing

Question 19

What is targeted PCR amplification?

Answer 19

16s rRNA, bacteria done more in bacteria than eukaryotes

Internal transcribed spacer (ITS), 18S rRNA eukaryotes

Question 20

What is 16S ribosomal RNA?

Answer 20

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

Question 21

What is the structure of 16S rRNA?

Answer 21

1542 base pairs with 9 variable regions and alternating conserved regions

Question 22

What do variable regions determine?

Answer 22

They determine the phylogenetics (which bacteria is present)

Question 23

Summarise the workflow of 16S targeted PCR amplification

Answer 23

Sample collection
DNA extraction 
16S PCR amplification 
Sequencing 
Analysis

Question 24

What can sequences in the 16S rRNA determine?

Answer 24

It can determine how many bacterial species are in the sample

Question 25

Describe briefly the changes in gut microbiota by age

Answer 25

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

Question 26

Which variable region is commonly used for sequencing?

Answer 26

V1 - V2 and V3 - V4

Question 27

What factors are considered when choosing a variable region?

Answer 27

- Phylogenetic signal | - Amplicon length

Question 28

What is the phylogenetic signal?

Answer 28

The genetic tree depending on the area being studied.

Question 29

What is the amplicon length?

Answer 29

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

Question 30

What is long read technology?

Answer 30

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

Question 31

What is 16S targeted PCR amplification sensitive to?

Answer 31

Contaimination from the environment, operator or reagents.

Question 32

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

Answer 32

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.

Question 33

How can potential contamination be mitigated?

Answer 33

- Randomise samples - Note batch numbers of reagents - Sequence negative controls

Question 34

What does the choice of variable region determine?

Answer 34

The resolution

Question 35

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

Answer 35

Introduce noise

Question 36

Describe the whole genome shotgun workflow

Answer 36

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.

Question 37

How is samples enriched without amplification pre-extraction?

Answer 37

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

Question 38

How are samples enriched without amplifcation post extraction?

Answer 38

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

Question 39

Benefits and Cons of Targeted 16S PCR amplification

Answer 39

Assess taxanomic diversity in sample | Biased, only bacteria

Question 40

Benefits of WGS sequencing

Answer 40

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