TC - Microbiome Tools

Question 1

What is a recent development that has made microbiome studies more accessible?

Answer 1

Substantial cost reduction for DNA sequencing

• zMicrobiome studies based on DNA sequencing are now affordable for many researchers worldwide.

Question 2

How are microbiome studies conducted across research fields? (2)

Answer 2

Highly similar across research fields

• The main difference is in initial sample preparation
• Methods (e.g., advanced microscopy, DNA, RNA, protein, or metabolite analyses) can be applied to various organisms and environments

Question 3

What is the starting point for many microbiome studies? (2)

Answer 3

Total-community DNA extraction

• Required for amplicon sequencing and metagenomics
• High-quality DNA extracts are crucial

Question 4

How is DNA sequencing performed in microbiome studies?

Answer 4

High-throughput sequencers produce millions of reads from total-community DNA extracts

Question 5

What is amplicon sequencing? (2)

Answer 5

Also known as metabarcoding

• A common tool to study microbiome composition
• Indexing PCR barcodes samples before pooling and sequencing

Question 6

What is metagenomics? (3)

Answer 6

• Sequencing of total-community DNA extracts without prior amplification
• More challenging data analysis compared to amplicon sequencing
• Allows assessments of functional repertoires at the gene level (no expression information)

Question 7

What are three other approaches in microbiome studies?

Answer 7

1) Metatranscriptomics – Based on total-community RNA extracts, reverse transcribed and sequenced

2) Metaproteomics – Based on total-community protein extracts, proteins separated and analysed using LC-MS

3) Metabolomics – Based on metabolite analysis, mostly using MS techniques

Question 8

Example for a metaproteomics approach (3)

Answer 8

• Studies based on metaproteomics allow to assess functioning at the protein level.
• The achievable depth is far lower than with metagenomics – therefore it only describes prevalent members of the microbiome.
• Limited availability of reference databases.

Question 9

How are proteins processed in metaproteomics studies? (3)

Answer 9

• Proteins are separated on two-dimensional gels
• Sequentially subjected to LC-MS analysis
• Identifies 10,000+ peptides compared to 10,000,000+ DNA sequences in metagenomics

Question 10

Why is metadata important in microbiome studies? (3)

Answer 10

• Researchers deposit DNA, RNA, protein sequences, and metadata in public repositories
• Ensures reproducibility and allows for re-analysis and inter-study comparisons
• Supports upcoming AI-based approaches

Question 11

What role does bioinformatics play in microbiome studies? (2)

Answer 11

• High-performance computers process millions of data points simultaneously
• Most researchers spend more time with data analysis than with wet lab work

Question 12

What are examples of bioinformatics tools? (3)

Answer 12

1) Network analyses – Visualises interactions within microbiomes

2) Beta diversity analyses – Shows similarities and dissimilarities between microbiomes

3) Heatmaps – Visualises prevalence or activity in microbiomes