24 March 2014

ddRADseq, Phusion PCR, and gut microbes

Next post Previous post

ddRADseq PCR amplification

Awesome open science initiative. Would make a great teaching opportunity.

In general, this is a pretty cool open science platform: Synbiota

From Twitter, John Edmonstone, a former black slave that taught taxidermy to Charles Darwin!

Bio Seminar

Jake Russell: Microbial profiling of gut symbionts of ants

  • symbionts key for use of poor diets; wood, sap, blood
  • Rhizobiales found in ants that have switched to herbivorous diet
  • Entomoplasmatales in predatory army ants
  • Within Cephalotes, one strain per individual, but multiple strains per colony
  • long-lived symbioses in existence for millions of years

My question: how are the mutualisms (assuming actually mutualistic) maintained? In legume - rhizobia and fig - fig wasps, sanctions (or partner fidelity) can occur on ‘organ’ level. How is this done for the whole gut? Jake suggested that it might occur at colony level. That is…‘cheater’ strains will be weeded out through expression in individuals…does this make sense? Need to think about and/or model it…

Also pointed out in talk that can use ‘blank’ sample in Illumina sequencing to quantify contamination. Doh! Should definitely do this for RADseq.


The final stretch!

PCR amplification

PCR for each size selected band (200, 300, 400) for each library (JSG001, JSG002).

Diluted stock PCR primers 2x to get 10uM working stock solutions.

Shared P1 for both libraries.

  • JSG001 amplify with P2_1
  • JSG002 amplify with P2_2

Used all 10ul of Dynabead-purified sample in PCR, as according to Dynabeads FAQ can use straight in PCR

For each sample, 20 ul reaction volume

Reagent Volume (ul)
DNA 10
HF Buffer 5
10mM dNTPs 0.4
PCR1 1
PCR2_X 1
DNA Polymeras e 0.2
H20 2.4

Made a seperate master mix for each PCR2 primer (one for each library). Ran PCR

Step Cycles Temp(c) Time (sec)
Initial denaturation 1 98 30
Denaturation 8 98 10
Annealing 8 65 10
Extension 8 72 15
Final extension 1 72 5 min

With the annealing temp from Yihong’s protocol with Pogos.

AMPure purification of the PCR-amplified product.

Eluted in 35ul.

Sent 2ul for Bioanalyzer, and will run a sample on Qubit tomorrow.


Gustafson, D.J., Major, C., Jones, D., Synovec, J., Baer, S.G. & Gibson, D.J. (2014). Genetic Sorting of Subordinate Species in Grassland Modulated by Intraspecific Variation in Dominant Species. PLoS ONE, 9, e91511.

  • how does intraspecific genetic variation in dominant species affect genetic structure of subordinate species?
  • community assembly experiment. cultivars or local ecotypes of Andropogon gerardii, Sorghastrun nutans, Schizachyrium scoparium
  • sif. differences in genetic structure of Chamaecrista fasciculata and Silphium integrifolium between cultivars and local ecotypes of dominant grasses
  • positive relationship between dominant and subordinate genetic diversiyt
  • “filtered through competitive exclusion of subordinate species genotypes by dominant species during community assembly”

Creative Commons Licence
This work is licensed under a Creative Commons Attribution 4.0 International License.