Techniques in microbiome studies

Question 1

Microbome

Answer 1

The microorganisms in a particular environment (including the body or a part of the body) or the combined genetic material of the microorganisms in a particular environment

Question 2

microbiota

Answer 2

The ecological community of commensal, symbiotic and pathogenic microorganisms found in and on all multicellular organisms studies to date from plants to animals (not walls, desks, and rocks).

Question 3

16S rRNA Targeted Amplicon Sequencing

Answer 3

The 16S rRNA genes from a particular sample are amplified from a sample and sequenced. This provides a look at which bacteria are inhabiting an environment.

Question 4

Metagenomics

Answer 4

All of the genetic material from an environment is sequenced. This provides a look at the functional potential of a microbial community with little or no information on individual organisms.

Question 5

Single-cell Genomics/ Read Cloud Sequencing

Answer 5

Sequences the genetic material from a microbial community either one cell at a time or after individual chromosomes have been labelled. This provides detailed genomic information about the most abundant individual organisms in a particular community.

Question 6

16S rRNA gene

Answer 6

• 9 hypervariable regions that are useful for the phylogenetic identification of bacteria, and therefore sequencing the 16S rRNA gene is a common method of bacterial identification
• 1542bp
• reliable molecular clock
• 1-16 16S rRNA genes
• NOT present in eukaryotes
• has slow evolution, so it can be linked to taxonomy

Question 7

rarefaction curves

Answer 7

X

Question 8

metagenomics technique

Answer 8

-In a metagenomics study you cut all of the DNA from a entire community up into small pieces and sequence it
Then you try to put it back together…
-Will tell you a lot about the functional potential of a microbial community, depending on how well you put it back together
-It will tell you nothing about which bacteria do what.

Question 9

Mothur

Answer 9

Mothur will not process sequence reads that don’t (almost) fully overlap
If the sequencing was done poorly, the program Mothur cannot process it, so seeing that the authors used Mothur is an indication that the sequencing was done well

Question 10

QIIME

Answer 10

Does not require that reads fully overlap and is recommended if you choose primers that don’t fully overlap forward and reverse reads
If the sequencing has a lot of errors QIIME will still produce a result
Good papers use QIIME, but if this program was used it’s not a direct indication that the sequencing was done well

Question 11

things to be critical of in metagenomic sequencing

Answer 11

1. Poor sequencing strategy
2. Confounded sample collection
3. DNA extraction
4. Data processing
5. Small sample sizes