The metagenome

Question 1

What is Metagenomics

Answer 1

the study of genetic material recovered directly from environmental or biological systems/compartments
- gives unbiased view of taxonomic diversity in a sample

Question 2

What is Microbiota

Answer 2

the combination of organisms in the compartment

ecological community of commensal and pathogenic microorganisms including bacteria, archae, protists, fungi and viruses

Question 3

What is Microbiome

Answer 3

microbiota + “theatre of activity” (which is the other biological components such as peptides, lipids, toxins etc)
the collective genomes of microorganisms in communities

Question 4

Taxonomic Diversity

Answer 4

The number and relative abundance of species in a community.

Taxonomic diversity varies by body site

Question 5

What have changes in human microbiome been associated with?

Answer 5

multiple human illnesses e.g.:

• IBS
• depression
• cancer

Question 6

Which microbiome can be used to classify individuals as lean or obese?

Answer 6

Gut microbiome (>90% accuracy)

Question 7

Which infection affects stool microbiome?

Answer 7

Clostridium difficile infection (CDI)

• stool microbiome infected with this is quite different from a healthy stool microbiome
• CDI has greater effect on stool microbiome than host genetic factors

Question 8

Restoration of healthy stool microbiome after CDI

Answer 8

Faecal microbiota transplant

- rapid restoration to healthy state

Question 9

What are the technological approaches carried out in Metagenomics?

Answer 9

1) Targeted PCR Amplification
> relies on 16S ribosomal RNA for bacteria

2) Whole Genome Shotgun Sequencing

Question 10

16S Targeted PCR Amplification

Answer 10

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

The gene for a 16SrRNA is split into variable regions and has conserved regions

• Variable regions are the parts that can be used to determine the different species that are present in a sample - contain the phylogenetic signals

Question 11

Steps of 16S Targeted PCR Amplification

Answer 11

1) Collect bacterial sample
2) Extract DNA from that sample
3) Perform a 16S PCR amplification
4) Put that on a sequencing machine
5) Sequences generated which match to the products we amplified which should match to the actual bacterial content of the original sample

Question 12

Data Analysis of 16S Targeted PCR Amplification

Answer 12

Sequences generated are compared to databases of 16S genes from different bacterial species to identify the species and abundance of species in the sample

This abundance type measurement is converted into a graph, with colours representing different bacterial species

Question 13

What is used to generate data from 16S Targeted PCR Amplification?

Answer 13

Software tools e.g.:

• QIIME2
• Mothur
• DADA2

Question 14

Problem with sequencing 16S gene

Answer 14

It is 1500 bases long and therefore you can’t sequence the whole gene on “short read sequencing machine”.

Therefore, only particular regions are sequenced e.g. V1-V2, V1-V3, V3-V5, V4

However, new long read platform sequencing machines are able to sequence the whole 16S gene (V1-V9)

Question 15

How do we choose which variable regions to sequence in 16S Targeted PCR Amplification?

Answer 15

Choosing variable regions is based on: depends on your experiment

• phylogenetic signal
• amplicon length

Question 16

Controls in 16S Targeted PCR Amplification

Answer 16

16S rRNA gene is found in all bacteria which makes it very sensitive to contamination by:

• operator
• environment
• reagents

*especially important for low biomass samples

Question 17

Kitome - how do we avoid contamination?

Answer 17

Avoids contamination by:

• randomise samples to avoid bias
• note batch numbers of reagents
• sequence negative controls (e.g. sequencing water could produce bacterial samples)

Question 18

What determines resolution at which you can identify the bacteria?

Answer 18

the choice of the variable region

*will not get good resolution below the genus level hence less reliable

Question 19

What determines resolution at which you can identify the bacteria?

Answer 19

the choice of the variable region

*will not get good resolution below the genus level hence less reliable

Question 20

What enables full length 16S sequencing?

Answer 20

New long read technology enables full length 16S sequencing through:

• PacBio
• Nanopore

Question 21

Disadvantage of long read technology

Answer 21

higher error rates of long read technology can introduce noise

Question 22

Whole Genome Shotgun Sequencing

Answer 22

Same concept as 16S PCR amplification as you take a sample collection with mixture of bacteria and extract DNA.

However, the whole genome is sequenced as opposed to the 16S gene and build an assembly.

Question 23

Data Analysis of Whole Genome Shotgun Sequencing

Answer 23

Because the entire genome is sequenced, many sequence reads are generated from the many genes sequenced, and these can be assembled to see how they all join up:

• phylogenetic tree constructed, taxonomic diversity analysed and relative bacterial abundance measured
• can carry out gene predictions and identify bacterial metabolic pathways which may be present in the context of conditions and diseases (not possible in 16S)

Question 24

Disadvantage of whole genome shotgun sequencing

Answer 24

no amplification step like in 16S PCR amplification, meaning:
- patient host cells are often in excess as no amplification step to enrich for bacterial DNA
= end up sequencing the whole patient DNA

Sample dependent, typical yields of contaminating human reads:
> Faecal: <10% human reads
> Saliva, nasal, skin: >90% human reads

Question 25

How to enrich bacterial sample so that there is less patient DNA without amplification?

Answer 25

Through pre-extraction or post-extraction:

PRE-EXTRACTION

• differential lysis (break down) of mammalian cells
• enriches for intact microbial cells
• however, introduces potential bias towards gram-positive bacteria

POST-EXTRACTION

• enzymatic degradation of methylated nucleotides targets mammalian DNA
• bias against AT rich bacterial genomes

Question 26

16S PCR amplification vs WGSS

Answer 26

Both:
-asses taxonomic diversity in a sample

However, 16S Targeted PCR amplification is biased (only bacteria), whereas WGSS is unbiased (all microorganisms)

Question 27

differences between targeted 16S PCR amplification and whole genome shotgun sequencing

Answer 27

16S PCR = access taxonomic diversity in sample BUT is biased bc only works on bacteria

whole gnome = also accesses taxonomic diversity, AND is unbiased, can work on all microorganisms. and it can access composite gene functions in the sample

they both have pretty much same steps