Lec 17 - The human microbiome for life

Question 1

How are humans holobionts?

Answer 1

More bacterial human cells (25 trillion vs 38 trillion)

Question 2

What does microbiota mean?

Answer 2

The MCOs present in a specific site

Question 3

What does microbiome mean?

Answer 3

Microbial community that occupies a well-defined habitat; including the collective genome contained within the microbiota

Question 4

What does prebiotic mean?

Answer 4

Substrate selectively used by host MCOs conferring a health benefit

Question 5

What does probiotic mean?

Answer 5

Live MCOs that when administered in adequate amounts confer a health benefit on the host

Question 6

What is a biome?

Answer 6

Well defined habitat with distinct bio-physio-chemical properties

Question 7

What is a microbiome?

Answer 7

Microbiota + theatre of activity (structural elements, secreted molecules, nucleic acids, environment where they live)

Question 8

What was the great plate anomaly?

Answer 8

Most bacteria seen under microscope can’t be cultured in lab

Question 9

Briefly describe how gene amplicons/16S rRNA gene sequencing is done

Answer 9

• NGS sequencing of all bacteria in sample via conserved region primers
• Sequence hyper and variable genes to identify bacteria
• Process
  1. Isolate sample DNA
  2. PCR 16s rRNA gene
  3. NGS
  4. Bioinformatics
  5. Count assigned numbers of sequence variants to find relevant abundance

Question 10

What are the pros and cons of gene amplicons/16S rRNA gene sequencing?

Answer 10

Pros = full length sequencing, species identification, quick, cheap, lots of info, low biomass, use with samples contaminated with host genome

Cons = limited to Illumina (200-300 bases) which restricts to specific regions = genus level identification, primers, false positives in low biomass

Question 11

What is taxonomic assignment and what are the 2 methods used in gene amplicons/16S rRNA gene sequencing?

Answer 11

• Family/genus/species level depending on conserved regions
• Operational taxonomic units (OTUs) = clustered similar sequences then use representative for each set = lose detail
• Amplicon sequence variant (ASV) = each sequence variant distinguished with less clustering

Question 12

Briefly describe shotgun metagenomics and the pros and cons

Answer 12

• Fragment and sequence all DNA present in sample using NGS
• Compare fragments against databases and assemble into genomes to find genes and their functions
• Pros = powerful, lots of info about community and functions, species/strain level identification, use for unculturable
• Cons = too much data, contamination with human DNA = wasted sequencing = expensive, time consuming analysis

Question 13

How is RNA measured and what are the pros and cons of this method?

Answer 13

• Metatranscriptomics
• Pros = identify live microbes, evaluate activity, transcript level responses
• Cons = complex collection and analysis, expensive and complex sequencing, contamination by host RNA

Question 14

What is culturonomics and what are the pros and cons?

Answer 14

• High throughput culturing using broad range conditions
• Pros = high throughput, targeted, provides isolates
• Cons = expensive, labour, influence by media conditions

Question 15

Describe how taxonomic composition barplots work. What is the main issue with them?

Answer 15

• Compares mean relative abundance against samples basically bar chart with sections in each bar
• Issue = uses mean which isn’t a representative of all samples

Question 16

Describe how taxonomic composition violin plots work including the following features :
- Width of each plot
- Dotted trend line
- White box in each plot
- Vertical lines in each plot
Why are they better than taxonomic composition barplots?

Answer 16

• Shows distribution of relative abundance across samples
• Width of each plot = number of individuals
• Dotted trend line = mean relative abundance
• White box in each plot = median relative abundance
• Vertical lines in each plot = percentiles
• Better bc tells you more about distribution of values across any time point

Question 17

What is biodiversity and how is it measured?

Answer 17

Variety within and among life forms in sample/ecosystem/landscapes measured by:
1. Richness = number of groups/OTUs/ASVs
2. Evenness = proportions of each of the different species/strains

Question 18

What is alpha and beta diversity?

Answer 18

• Alpha = measure of mean diversity within a sample eg high alpha diversity = high richness and evenness
• Beta = measure of diversity between samples eg low beta diversity = samples share same species at similar proportions

Question 19

What 2 methods are used to visualise beta diversity?

Answer 19

1. Distance matrices = compare samples by summarising pairwise differences to make hierarchical clustering (phylogenetic tree looking thing)
2. Principal component analysis (PCA) = samples together within one circle/colour have low beta diversity and samples further apart have high; not as accurate (like those cytokine chart thingos)

Question 20

What is differential abundance testing?

Answer 20

• Identify different bacterial abundances between groups
• Compares every taxa between healthy and diseased
• Huge data created by multiple tests with a cutoff P value
• Requires specialised statistic tests to minimise false positives generated by using so many tests