Microbiomics/microbiota II

Question 1

What are the steps of the process of 16S rRNA analysis? (4)

Answer 1

• DNA isolation
• Mostly targeted sequencing using 16S rRNA amplicon
• Sequencing (Illumina)
• Analysis

Question 2

What is the target gene of choice in 16S rRNA sequencing? What is the advantage of this?

Answer 2

16S rRNA ribosomal subunit -> present in all species

Question 3

What do the variable regions determine in 16S rRNA sequencing?

Answer 3

Phylogeny

Question 4

What is the gene length in 16S rRNA sequencing?

Answer 4

~ 400 bp

Question 5

What are the steps of the sequencing process of the 16S gene?

Answer 5

• Amplification of 16S gene (PCR)
• Sequencing

Question 6

What are the steps of amplification of 16S gene through PCR? (2)

Answer 6

• Select for 16S gene
• Add sequencing adaptors

Question 7

What are the steps of the analysis pipeline in 16S sequencing? (3)

Answer 7

• Reads are grouped based on sequence homology
• Denoising
• Taxonomy is assigned

Question 8

To what does analysis of 16S genes lead?

Answer 8

Amplicon sequence variants (ASV) -> operational taxonomic units (OTU)

Question 9

What is an OTU?

Answer 9

Virtual species/strain

Question 10

What is an OTU based on?

Answer 10

Clustering of homologous sequence reads

Question 11

What kind of measure is number of reads per OTU?

Answer 11

Abundance