metagenome

Question 1

What is metagenomics?

Answer 1

• study of whole sequence in cell (overall view of gene content in sample)
• organisms do not live in isolation so genetic material is recovered from environmental or biological systems.
• unbiased view of taxanomic diversity in a sample
• not limited to ability to culture (some cant be cultured)

Question 2

What is microbiota?

Answer 2

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

Question 3

What is microbiome?

Answer 3

a characteristic microbial community occupying a reasonably well-defined habitat which has distinct physio-chemical properties. The term thus not only refers to the microorganisms involved but also encompasses their theatre activity.

Question 4

What is human micro-biome?

Answer 4

• taxonomic diversity varies by body site
• unique to each individual (even twins)
• changes in micro biome can be associated with disease (IBS, cancer, depression)
• can classify humans as lean/obese (90%) accuracy

Question 5

define the 4 ‘omics’

Answer 5

1. genomic = whole cell gene count
2. transcriptomics = whole cell gene expression
3. proteomics = whole cell protein content.
4. metabolomics = whole cell metabolite.

Question 6

how can we use human microbes to identify disease?

Answer 6

• microbiome unique to each individual, even between twins.
• changes in the microbiome have been associated with multiple human illness, eg: irritable bowel syndrome, depression, cancer
• help classify individuals as lean or obese with >90% accuracy
• early life microbiomes linked to development of allergic conditions, eg. asthma

Question 7

What is an example of disease which can be identified by looking at stool microbiome and how can you treat it?

Answer 7

• clostridium difficile infection (CDI)
• CDI has greater effect on stool than host genetic factors
  cure:
• faecal microbiota transplant is able from healthy patients
  -restoration of the stool microbiome to that of healthy state is rapid following transplantation.

Question 8

what are the steps to 16S targeted PCR amplification workflow?

Answer 8

1. sample collection
2. DNA extraction
3. 16S PCR amplification
4. sequencing
5. analysing

Question 9

What is the genetic make up of 16S ribosomal RNA?

Answer 9

• 16S ribosomal RNA is a component of 30S subunit of prokaryotic ribosome.
• about 1500 bases long
• separated into 9 variable regions
• has conserved regions too

Question 10

What are the 5 common variable regions used for PCR?

Answer 10

V1- V2
V1- V3
 V3 - V4
V3- V5
V4

Question 11

Which phylogenetic signal tree separates species well?

Answer 11

=>V1- V2 separates species well

=> V4 doesn’t distinguish species well

Question 12

How does amplicon length affect quality of data?

Answer 12

V1- V3 only partly overlaps
V1- V2 completely overlaps with V4
when variables overlap the quality of data is better.

Question 13

What is the advantage of high throughput sequencing?

Answer 13

• the sequence length increases
• meaning we can genome the entire sequence
• much greater resolution.

Question 14

Why must we use controls?

Answer 14

• method targeted PCR amplification is very sensitive to contamination so
• must control for contaminations coming from reagent used.

Question 15

How do we mitigate potential contamination?

Answer 15

• randomise samples
• note batch numbers of reagent
• sequence negative control

Question 16

compare the 2 technological approaches : targeted 16S PCR amplification and genome sotgun sequencing?

Answer 16

targeted 16S PCR :
=> asses taxanomic diversity in sample
=> biased, only bacteria

whole genome shotgun:
=> assess taxonomic diversity in sample
=> assess composite gene functions in sample
=> unbiased, all micro-organisms

Question 17

What are problems with whole genome shotgun workflow?

Answer 17

=> host cells often in excess in the sample
=> no amplification step to enrich for bacterial DNA
=> sample dependent, typical yields of contaminating human reads :
feacal : <10%
saliva, nasal, skin samples : > 90% human reads

Question 18

How do we enrich genome without amplification?

Answer 18

1. pre-extraction
   - differential lysis of mammalian
   - enrich for intact microbial cells
   - potential bias towards gram -positive
2. post -extraction
   - enzymatic degradation of methylated nucleotides targets mammalian DNA
   - Bias against AT rich bacterial genomes

Question 19

What are 3 different meta-genomics applications?

Answer 19

• environmental
• animal
• clinical diagnostics

Question 20

What are features of the environmental genome shotgun sequencing of the saragasso sea(2004)?

Answer 20

• 1.045 billion base pairs sequenced
• elucidate the gene content, diversity, and relative abundance of the organisms
• estimated to derive from 1800 genomic species
• identified 148 previously unknown bacterial phylotypes
• identified over 1.2 million previously unkown genes

Question 21

What are features of animal microbiomes (Rumen microbiomes)?

Answer 21

• first of 4 chambers of cows stomach
• contains a mix of bacteria and other organisms which ferment complex carbohydrates to produce short chain fatty acids
• generated 6.5 terabases short and long read sequence ruminant cattle
• assembled 4,941 genomes including 3 whole chromosome assemblies rumen bacteria.

Question 22

how can metagenomics be used clinically for diagnosis?

Answer 22

• gold standard is to culture isolate and then identify using matrix assisted laser desorption/ionisation (MALD)
• many organisms cannot be cultured
• can identify hard culture organisms in patient samples e.g neuroleptospirosis in 14 y/o critically ill boy with meningoencephalitis
• identify antibiotic resistance repertoires directly from clinical samples.
• potential to develop diagnostics based on differences in micro biomes.

Question 23

How is clinical metagenomics used in public health?

Answer 23

• infection control and outbreak management

- surveillance of antimicrobial resistance in the food supply.