15. THE METAGENOME

Question 1

What is Metagenomics?

Answer 1

• Metagenomics is the study of genetic material taken directly from environmental or biological samples/compartments

Question 2

What’s the difference between microbiome & the microbiota?

Answer 2

• Microbiome is also known as the metagenome, it includes micro-organisms such as bacteria, prokaryotes as well as the genes they contain & the environmental factors influencing them
• The microbiota refers to the combination of micro-organisms within an environment such as protists, fungi, bacteria etc.

Question 3

What are some examples of human & environmental microbiota?

Answer 3

• ENVIRONMENTAL - soul microbiome, deep sea microbiome

- HUMAN - gut microbiome, skin microbiome, oral microbiome

Question 4

What can differences in the human microbiome tell us?

Answer 4

• The human microbiome varies between individuals and is unique for each individual
• Significant differences in the microbiome have been associated with various diseases such as depression, cancer, IBS
• The gut microbiome can also be used to classfify individuals as obese or lean with greater than 90% accuracy

Question 5

What’s the effect of Clostridum Difficil on the stool microbiome?

Answer 5

• A clostridum difficil infecion can change the stool microbiome & make it vary significantly from the healthy microbiome
• A faecal transplant can help restore the microbiome

Question 6

What are the two sub-units of prokaryotes & eukaryotes & what do they consist of?

Answer 6

EUKARYOTES:
- 60S = 5S & 28S rRNA
- 40S = 18S rRNA
PROKARYOTES:
- 50S = 5S & 23 rRNA
- 30S = 16S rRNA

Question 7

What is 16S rRNA PCR targeted amplification?

Answer 7

• 16S rRNA is a component of the 30S small subunit of prokaryotic ribosomes, found in all bacteria
• Targeted 16S rRNA amplification assesses taxonomic diversity in a sample
• However, it’s biased as it only looks at bacteria

Question 8

*What are the steps involved in Targeted PCR 16S rNA amplification?

Answer 8

1. Sample collection
2. DNA extraction
3. 16S rRNA PCR amplification
4. Sequencing
5. Analysis

Question 9

Name 3 analysis softwares for 16S rRNA targeted PCR amplification

Answer 9

1. MOTHUR
2. QIIMEZ
3. DADA2

Question 10

What are the two regions of the 16S rRNA?

Answer 10

1. Conserved region
2. Variable region (e.g V1)
   - The variable region can be sequenced & can tell us about differences

Question 11

What two factors need to be considered when choosing the variable region of the 16S rRNA?

Answer 11

1. Phylogenetic signal
2. Amplicon length
   - If two sequences overlap, the errors in the overlap will be corrected
   - Two short sequences won’t overlap as much, so not all the errors will be corrected
   - Two longer sequences will overlap completely & the errors will be corrected

Question 12

How can the sequencing machine affect the read length?

Answer 12

• There are two types of sequencing machine which can be used in 16SrRNA targeted PCR amplification:
  1. Short read - Roche 54 (700bp), Illumina Miseq (2 x 300bp), Illumina Next seq & Hiseq (2 x150bp)
  2. Long read - PacBio (10Kb), ONT MINON (15Kb)

Question 13

What is the kitome?

Answer 13

• The kitome refers to the microbiome which has been contaminated

Question 14

How can we avoid potential contamination of samples in 16S rRNA targeted PCR amplification?

Answer 14

1. Randomise samples to even the samples out or so that a particular sample has less of an overall effect
2. Note batch numbers to identify a bad batch
3. Sequence the negative control so that any contamination can be accounted for

Question 15

**What is Whole genome shotgun sequencing?

Answer 15

• Whole genome shotgun sequencing can assess taxonomic diversity & composite gene functions in a sample
• WGS is unbiased as it looks at all microrganisms

Question 16

What are the two outcomes of WGS sequencing?

Answer 16

1. Produce an ASSEMBLY - to show taxonomic diversity

2. BINNING - sequences can be compared to taxonomic sequences & can be grouped into taxonomic groups

Question 17

Name 3 softwares used by WGS sequencing

Answer 17

1. MG-RAST
2. METAPHLAN
3. KRAKEN2

Question 18

Why is there no amplifications step in WGS sequencing?

Answer 18

• Amplification isn’t needed to enrich the bacterial DNA because the host cells are often in excess in the sample
• However, this is sample dependent
• Saliva, nasal & skin samples have a low biomass but produce greater than 90% reads
• Whereas faecal samples have a high biomass but produce less than 10% reads

Question 19

*Give two ways in which we can enrich the sample without amplification in PCR?

Answer 19

1. PRE-EXTRACTION - Differential lysis of mammalian cells to enrich microbial cells
   - Possible bias for gram bacteria
2. POST-EXTRACTION - Enzymatic degradation of methylated nucleotides targets mammalian cell
   - Possible bias for AT rich bacteria

Question 20

Give three applications of WGS sequencing

Answer 20

1. Animal microbiome - Rumen (chambers of cows gut) can be sequenced & used to reassemble whole genome
2. Environmental - can sequence an area to identify unknown phylotypes & genes
3. Clinical - Potential to develop diagnostic based on differences in the microbiome