TC - Tools for Microbiome Analysis I

Question 1

What is the microbiota and what are its constituents? (5)

Answer 1

The microbiota comprises all living members forming the microbiome.

• Bacteria
• Archaea
• Fungi
• Algae,
• Small protists

(Note: Viruses, plasmids, and mobile genetic elements are part of the microbiome but not the microbiota.)

Question 2

What is a Holobiont? (4)

Answer 2

• An entity that includes the host and its microbiome
• The host genome is complemented by the microbiome’s metagenome
• Combined genetic information is known as the hologenome
• Hosts co-evolve with their microbiome

Question 3

Why is understanding microbial communities important? (3)

Answer 3

• Microorganisms interact within complex microbial communities, rarely existing in isolation
• Better understanding can lead to precision treatments
• Shaping microbiomes can improve human, animal, and plant health

Question 4

How are microbiomes impacting the retail sector?

Answer 4

Microbiome-related products have been introduced as researchers and the public recognize their potential

Question 5

How are microbiomes analysed? (4)

Answer 5

• Advanced microscopy techniques (e.g., FISH-CLSM)
• Advanced cultivation techniques
• High-throughput analyses of DNA, RNA, and proteins
• Most current analyses are based on DNA sequencing

Question 6

Why are microbiomes analysed with different tools?

Answer 6

DNA-based microbiome analyses allow us to holistically describe microbiome compositions (who is there), but not functions (what are they doing).

• Analyses based on RNA and proteins allow us to better capture functions of a microbiome.

Question 7

What is FISH-CLSM and how does it work? (3)

Answer 7

Fluorescence in situ hybridisation (FISH) allows labeling specific microorganisms with fluorescent probes

• Confocal laser scanning microscopy (CLSM) visualises the labeled microbes in complex communities
• FISH-CLSM can simultaneously analyse up to seven microbial groups

Question 8

What are features of FISH probes? (2)

Answer 8

• These probes are similar to PCR primers (DNA-based, short oligonucleotides) but are additionally bound to fluorescent dyes.
• They mostly target rRNA in microorganisms and allow visualisation with a confocal microscope.

Question 9

Microbiome analyses based on FISH-CLSM (2)

Answer 9

• Visualisation of bacteria colonising plant roots (Brassica napus). Individual bacteria are visible and enriched around root hairs.
• Visualisation of bacteria colonising a lichen. Different bacterial colonies are visible as well as fungal hyphae and algae of the lichen host.

Question 10

What is Culturomics? (2)

Answer 10

• A method to culture microorganisms by using hundreds of different media types simultaneously
• Developed to increase the number of cultivable microorganisms

Question 11

What is DGGE and why has it been replaced? (3)

Answer 11

• Denaturing gradient gel electrophoresis (DGGE) was used to study microbial communities before 2010
• It was labor-intensive and low in reproducibility
• High-throughput DNA sequencing has replaced it due to its speed and precision

Question 12

How have advances in DNA sequencing benefited microbiome research? (3)

Answer 12

• New instruments generate millions of reads in hours
• Deep sequencing is required to holistically describe microbiomes – especially if they are complex.