The metagenome

Question 1

What is genomics?

Answer 1

Whole cell gene content

Question 2

What is transcriptomics?

Answer 2

Whole cell gene expression

Question 3

What is proteomics?

Answer 3

Whole cell protein content

Question 4

What is metabolomics?

Answer 4

Whole cell metabolite content

Question 5

What is metagenomics?

Answer 5

It’s the study of genetic material recovered directly from environmental or biological systems

• Gives an unbiased view of taxonomic diversity in a sample
• Not limited by ability to culture
• Overall view of gene content in a sample

Question 6

What is microbiota?

Answer 6

It is the ecological community of commensal and pathogenic microorganisms Includes: bacteria, archare, protists and fungi

Question 7

What is microbiome?

Answer 7

It is the collective genomes of the microorganisms in microbiota

Question 8

How is microbiomes to each individual and so what can we deduce?

Answer 8

Reminder= Microbiome is the collective genomes of micro-organisms in microbiota

Microbiome are unique to each individual (even in twins), so more to do with environmental factors

Changes in the gut microbiome have been associated with multiple human illnesses (IBS, depression, cancer)

• The gut microbiome can classify individuals as lean or obese with 90% accuracy.
• Early life gut microbiomes are linked to the development of asthma

Question 9

What can gut microbiome in individuals classify?

Answer 9

Gut microbiome can classify individuals as lean or obese with >90% accuracy

Question 10

Describe the human stool microbiome when infected with CDI

Answer 10

• Stool microbiome when infected with CDI (clostridium dificle) is different to a healthy stool microbiome
• CDI has a greater effect on the stool microbiome than host genetic factors
• Faecal microbiota transplant is able to cure CDI
  
  • Restoration of the stool microbiome to that of healthy state is rapid following transplantation

Question 11

What does CDI have a greater effect on?

Answer 11

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

Question 12

List the technological approaches used in metagenomics

Answer 12

• Targeted PCR amplification
  
  • 16S RNA bacteria
  • Internal transcribed Spacer (ITS), 18S rRNA eukaryotes
• Whole Genome Shotgun Sequencing

Question 13

What is 16s rRNA?

Answer 13

16s rRNA is a component of 30s small subunit of prokaryotic ribosome

Question 14

What do we use to diversify between species in targeted 16S PCR amplification?

Answer 14

We can use the variable region to identify the phylogenetics of different species present in a sample

• This is because the variable regions are conserved within a phylum (genetic group) but converge within a species
• These regions are used to seperate species based on these sequences

Question 15

Describe how 16S targeted amplification works?

Answer 15

• We collect a mixed sample collection
• Then carry out DNA extraction of all bacteria
• Afterwards we do 16s PCR amplification
• Sequencing
• Analysis

By matching sequenced samples, you are abel to identify how many species or genus is present in the sample. This can then be converted into data

Question 16

What factors do we take into account when choosing the variable regions (V1-V9) to choose in 16s PCR amplification?

Answer 16

• Phylogenetic signal - does the variable region contain enough information to seperate the species that you’re interested in seperating
• Amplicon length - how big can the PCR product be?

Question 17

How do you prevent contamination of a sample in 16S targeted amplification?

Answer 17

The method is very sensitive to the contamination from the environment, operator and reagents = this is important for low biomass samples

contamination can be avoided by:

1. Randomise samples avoids bias!!!!
2. Note batch numbers of reagents!!!!
3. Sequence negative controls e.g sequencing water!!

Question 18

Describe whole genome shotgun sequencing?

Answer 18

Has the same steps as 16s PCR amplification but the whole genome is sequenced instead of only 16s rRNA. = NO BIAS

These reads can be assembled, a phylogenetic tree is constructed and taxonomic diversity is analysed, the relative bacterial abundance can be looked at.

• By finding out which genes are present, we infer which metabolic pathway is present in the sample.
  
  • Comparing with other samples you are able to look for enrichment, decreasing metabolic pathways between different environments, niches

Question 19

What do we to do the data we obtain from whole genome shotgun technique and how can we use this data?

Answer 19

The sequence from the sequencing machine is reassembled to make sense -Can use the data to do a taxonomic diversity assessment -Can also use the data for gene prediction

Question 20

How do we enrich without amplification?(Pre extraction)

Answer 20

• Different lysis of mammalian cells
• Enrichs for intact microbial cells
• However potential bias towards gram positive bacteria

Question 21

How do we enrich without amplification?(Post extraction)

Answer 21

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

Question 22

How does 16S targeted amplification cope with hard reads?

Answer 22

• Choice of variable region determines resolution. It is less reliable below genus level
• Thus 16S is hard with long reads
  
  • New Technology enables full length 16S sequencing
    
    • PacBio + Nanopore
• However, higher error rates of long read technologies introduce noiseee. Development of this technology is ongoing.

Question 23

What is an issue with whole genome shotgun sequencing?

Answer 23

• Host cells are often in excess in the sample, no amplification step which enriches for bacterial DNA
• It is sample dependant, typical yields of contaminating human reads are:
  
  • Faecal: <10% human reads
  • Salivia, nasal, skin samples >90% human reads

The sample can be enriched so that there is less patient DNA/host cells without amplification through pre-extraction or post-extraction

Question 24

What is the difference between targeted 16S PCR amplification and whole genome shotgun sequencing?

Answer 24

• Targeted 16S PCR amplification
  
  • Assess taxanomic diversity in sample
  • Biased, only bacteria
• Whole genome shotgun sequencing
  
  • Assess taxanomic diversity in sample
  • Assess composite gene functions in sample
  • Unbiased, all micro-organisms