Metagenome

Question 1

What is genomics?

Answer 1

The whole cell content

Question 2

What are early life gut microbiomes linked with?

Answer 2

Development of allergic conditions such as asthma

Question 3

What is proteomics?

Answer 3

Whole cell protein content

Question 4

What is metabolomics?

Answer 4

Cell metabolite content

Question 5

How do you purify DNA?

Answer 5

Culture the organism in isolation

Question 6

Why in practise is DNA not able to be purified?

Answer 6

Organisms do not live in isolation
They are a complex mixture of species

Question 7

What is a microbiota and what does this include?

Answer 7

Ecological community of commensal and pathogenic microorganisms
Bacteria, archae, prostists, fungi and viruses

Question 8

What is a microbiome?

Answer 8

Collective genomes of the microorganisms in these communities

Question 9

What diseases have changes in the microbiome been associated with?

Answer 9

→ Cancer
→ Depression
→IBS

Question 10

What can gut microbiome classify?

Answer 10

individuals as being obese or lean

Question 11

What are early life gut microbiomes linked with?

Answer 11

Development of allergic conditions such as asthma

Question 12

What are the 4 human microbiomes?

Answer 12

Gut microbiome
Skin microbiome
Oral microbiome
Vaginal microbiome

Question 13

What can cure a clostridium difficile infection and why?

Answer 13

Stool transplant
The microbiome in CDI is different to a healthy microbiome

Question 14

Why is 16S targeted PCR used?

Answer 14

16S is a ribosomal component of the 30S subunit in prokaryotes

All bacteria have the 16S subunit

Question 15

Describe how 16S targeted PCR amplification works?

Answer 15

→ Sample (urine, blood, etc)
→ DNA is extracted from a mixed population of bacteria in the sample
→ 16S PCR amplification - the 16S gene amplifies
→ sequence the PCR

Question 16

Why are there biases in 16S PCR?

Answer 16

→ Some bacteria purify better than others
→ some sequences amplify better than others

Question 17

What is done with the sequences after 16S PCR?

Answer 17

Compared to a database of known 16S sequences

Question 18

What bacteria changes during the first year of life and how?

Answer 18

Actinobacteria drops after the first years of life

Question 19

What bacteria is present in babies when breastfeeding?

Answer 19

Bifidobacteria

Question 20

What 2 things do you need to consider when doing PCR on a bacterial variable region?

Answer 20

→ If it contains enough information to separate the genus
→ Amplicon length

Question 21

Why is sequencing a smaller region better?

Answer 21

The sequencer can read it twice and any errors will cancel out because the two sequences are combined into one region

Question 22

What are disadvantages to the 16S method?

Answer 22

→ It is sensitive to contamination
“Kitome” - reagents can have carry over contaminates

Question 23

What is the 16S method used for?

Answer 23

Low biomass sample

Question 24

How do you mitigate contamination?

Answer 24

→ Randomise the samples
→ Note batch numbers of reagents
→ Sequence negative controls

Question 25

What are the disadvantages of long reads?

Answer 25

→ They have higher error rates and introduce noise

Question 26

What is whole genome shotgun sequencing?

Answer 26

Sequencing all the genes not just the 16S

Question 27

How does whole genome shotgun sequencing work?

Answer 27

→ The entire genome is broken into small fragments of DNA → Short fragments are sequenced and assembled together by computer that find overlaps → You can assess the DNA taxonomically → Overlay it on pathways → Changes in metabolic pathways between species

Question 28

What are you looking for in whole genome shotgun sequencing?

Answer 28

Changes in metabolic pathways between species

Question 29

What are the 3 issues with whole genome shotgun sequencing?

Answer 29

→ Host cells are in excess → No amplification step to enrich for bacterial DNA → Sample dependent

Question 30

How do you enrich without amplification (pre extraction)?

Answer 30

→ Differential lysis of mammalian cells - bacteria have different cell walls → Put reagents to lyse the mammalian cells but not bacteria

Question 31

Why can there be a bias with lysing mammalian cells?

Answer 31

Bias towards gram +ve bacteria which have a thicker cell wall

Question 32

How do you enrich without amplificaiton (post extraction)?

Answer 32

→ Enzymatic degradation of methylated nucleotides which target mammalian DNA → Bias against AT rich bacterial genomes

Question 33

How is metagenomics used in diagnostic microbiology?

Answer 33

→ Culture isolate and then identify using matrix assisted laser desorption/ionization (MALDI) → Can identify hard to culture organisms in patient samples

Question 34

How can metagenomics be used in public health?

Answer 34

→ Infection control and outbreak management → Surveillance of antimicrobial resistance in the food supply

Question 35

How do you decide which variable to choose?

Answer 35

→ Phylogenetic signal- (shorter sample will correct mistakes as read twice/overlap). → Amplicon length- changes with intro of new sequencing platforms

Question 36

What are the two ways of assessing shotgun sequences?

Answer 36

1. Assembly 2. Binning

Question 37

What is binning?

Answer 37

Process of grouping reads or contains and assigning them to individual genome by comparing to databases

Question 38

Targeted 16S PCR amplification summary

Answer 38

→ Assess taxonomic diversity in sample → Biased, only bacteria

Question 39

Whole genome shotgun sequencing summary

Answer 39

→ Assess taxonomic diversity in sample → Assess composite gene functions in sample → Unbiased, all microorganisms