Approaches to soil + mehtods

Question 1

What is amplicon sequencing?

Answer 1

• Targeted approach: requires primers that select for a certain part of the genome
• Most commonly used based on 16S rRNA (bacteria) and for fungi 18S or ITS region
• Gives in-depth taxonomic identification of your bacteria;l or fungal community. If you want both bacteria and fungi everything has to be done twice.

Question 2

What are advantages of amplicon sequencing?

Answer 2

• Good for taxonomy (diversity metrics)
• Knowing who is present
• Much less bioinformatics compared to more advanced techniques

Question 3

What are disadvantages of amplicon sequencing?

Answer 3

• Primers are biased
• Dosen’t provide any functional information
• Identified species may not be active (DNA can be old or associated with dormant bacteria)

Question 4

What is the difference between amplicon and metagenomics?

Answer 4

Amplicon:

• Less computational
• More samples per run
• Deeper reader- longer DNA fragments characterize down to species easier

Metagenomic:

• Highly computational
• More expensive
• Shallow reads (smaller gene fragments) so taxa identification is more challenging
• Less biased
• Get all genes (including functional ones)

Question 5

What is cellular lipid analysis?

Answer 5

• Microbes have distinct phospholipid fatty acids (PLFAs) in their cell memebrane that can be used to classify microbes into coarse groups

Can obtain quantitative and relative abundance (bacteria and fungi)

Question 6

What are advantages of PLFAs?

Answer 6

Quantitative

some taxonomic resolution

Can be coupled with 13C assays to trace substrate use

Good approach for obtaining fungal: bacterial rations and biomass

Question 7

What are some disadvantages of PLFAs?

Answer 7

• Limited resolution
• Dead-end
• Labor intensive (8-12 samples in 3 days) and expensive

Question 8

What are methods to measure abundance?

Answer 8

• Plate counts
• Total microbial biomass : from extractions. Most common for sol scientists
• PLFA and q PCR (seperate fungal and bacteria abundances)

Question 9

What are methods to know total microbial biomass?

Answer 9

• direct count
• ## Chloroform-fumigation direct extraction (commonly used, relatively easy to use)

Question 10

What are functional assays?

Answer 10

Use when you want to know what an organism, population, community is doing to contribute to an ecosystemservice of concern.

Question 11

What are examples of functional assays?

Answer 11

• Decomposition
• Mineralization (CO2 respiration), Nitrogen mineralization
• Enzymes
• Growth rates
• Stable isotop probing

Question 12

What are tools to assess microbial activity in the field (respiration and GHG production=> CO2, N2O, CH4)?

Answer 12

• Manual gas sampling
• Automoatic chambers
• Eddy-covariance flux towers