The Metagenome

Question 1

What is metagenomics?

Answer 1

Metagenomics is the study of genetic material recovered directly from environmental or biological systems/compartments

Question 2

What are human microbiomes like?

Answer 2

Microbiome unique to each individual, even between twins
Changes in the microbiome have been associated with multiple human illnesses, e.g. Irritable Bowel Syndrome, depression, cancer.
Gut microbiome can classify individuals as lean or obese with >90% accuracy
Early-life gut microbiomes linked to development of allergic conditions e.g. asthma

Question 3

Describe the workflow of 16S targeted PCR amplification.

Answer 3

Starts with a sample collection process where we take a sample and break it up; separating the bacterial cells
This is followed by a DNA extraction event, where we separate all the DNA from all the bacterial species cells in the sample
We then do our 16S PCR amplification and we get different PCR products from each of those bacteria in the sample
We run those on our sequencing machine and we generate a sequence for each of those 16S genes
We then take these bacterial sequences and compare them to the known 16S database, with the three most common sequences being Greengenes, Silva and RDP
Then we can identify from which species they come from and their relative abundance

Question 4

Why do we have variable regions in 16S PCR amplification?

Answer 4

16S ribosomal RNA is component of 30S small subunit of prokaryotic ribosome
From the information above what that means is that we can only sequence part of the gene, a couple of hundred bases
Common regions used are V1-V2, V1-V3, V3-V4, V3-V5 and sometimes just V4

Question 5

How do we know which variable region to choose 16S targeted PCR amplification?

Answer 5

Phylogenetic signal
As you can see in V1-V2 the two types of bacteria are neatly separated whereas in V4 they are not so it really depends on use e.g. skin microbiota

Amplicon length:
V1-V3 amplicon is around 400 base pairs while the MiSeq is 2 by 300bp so they will only overlap to a certain percentage in the middle
What that means is, once you have assembled it together you get a 500bp product but any single point errors that sometimes you get in your sequence will only be corrected if the two amplicons overlap
Compare that to a V1-V2 or a V4 amplicon where the MiSeq will overlap everything means that there will be no single point errors

Question 6

What is a “kitome”?

Answer 6

“Kitome”- the reagents/contamination is carried through

Question 7

How do we mitigate potential contamination?

Answer 7

Randomise samples
Note batch numbers of reagents
Sequence negative controls

Question 8

What are the different ways we can perform whole-genome shotgun?

Answer 8

We can take out WGS shotgun sequences and we can form an assembly; putting the small shotgun sequences into a larger quantities here through various pieces of software
Another approach would be something called binning where we take out sequences, we compare to databases of taxonomic examples and we put those sequences into different bins based on which species/taxonomic group they belong to
Once we have our assembly we can do our taxonomic diversity; we can build an evolutionary tree showing the variation in the samples
But we can also use our assembly to predict genes, look at our genes and see which biochemical pathways are present in our sample

Question 9

Describe the whole-genome shotgun workflow.

Answer 9

Host cells often in excess in the sample so e.g. we are also sequencing human DNA
No amplification step to enrich for bacterial DNA
Sample dependent, typical yields of contaminating human reads:
- Faecal: <10% human reads
- Saliva, nasal, skin samples: >90% human reads
How to enrich without amplification?

Question 10

How do we enrich without amplification (in WGS)?

Answer 10

Pre-extraction

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

Post-extraction

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

Question 11

What are the capabilities of diagnostic microbiology?

Answer 11

• Gold standard is to culture isolate and then identify using matrix assisted laser desorption/ionization (MALDI)
  
  • Many organisms cannot be cultured
  • Can identify hard to culture organisms in patient samples e.g. neuroleptospirosis in a 14-year-old critically ill boy with meningoencephalitis
  • Identify antibiotic resistance repertoires directly from clinical samples