22 November 2013

DNA, extraction, and transcriptome

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Wed - Fri notes


Climate cascade

Lacy Chick in town from Tennessee after IBS conference.

Meeting to discuss Aphaenogaster project:

  • LC phytotron results for CTmax and CTmin
    • little evidence for lab acclimation within generation
    • CTmax and CTmin correlated with latitude, as well as thermal breadth (CTmax - CTmin)
    • less variation in CTmax than CTmin
    • more physiological constraints on CTmax than CTmin?
    • will morphological IDs be congruent with genetic relationships?
  • Pair phytotron results with Lacy’s field results from 2012
    • 55 sites from Gainesville FL to Millinocket ME
    • CTmax and CTmin from field site
    • consistent results with phytotron
  • Analysis
    • partial Mantel tests
    • spatial analysis: take top PCs from PCA on kinship matrix. use as continuous predictor with CTmax/min/thermal breadth as response
  • discussion of authorsip


Yesterday, test extraction to see if I can resolve last week’s failure to get DNA from Aphaenogaster. Set up two extractions

  1. 5 Aphaenogaster
  2. 5 Pogo

Treated identically.

Followed tissue homogenization protocol without RNAse A incubation and shorten air-dry time after centrifugation.

Genomic-tip DNA extraction. Saw DNA nice and clear at isopropanol precipitation step in both tubes, qualitatively more in the Aphaenogaster tube!

Eluted in 500ul Buffer AE


Sample ng/ul 260:280
Ap20131121-ali4 0.58 0.73
Ap20131121 3.3 1.2
Po20131121-ali4 -3.6 0.12
Po20131121 -0.2 -0.35

Initially concerning, but I eluted with 500ul so 3.3 ng/ul * 500 ul = 1650 ng DNA! This would be quite good extraction if correct. The Pogo extraction less encouraging. Method seems to be quite finicky!


Message from Mahesh that he updated VGN website with results of BLAST of the transcriptome set against itself:



Precipitated Ap DNA with 70% ethanol, rehydrated in 100ul Buffer AE. Nothing. argh.


In process of modifying scripts to generate transcriptome using all reads from both colonies as done by Mahesh, but using diginorm. Then map each sample individually to combined transcriptome.

Molecular Ecologist

Interview questions to Richard Lenski


Roux, J., Privman, E., Moretti, S., Daub, J.T., Robinson-Rechavi, M. & Keller, L. (2013). Patterns of positive selection in seven ant genomes. arXiv:1311.4706 [q-bio]. Retrieved November 21, 2013, from http://arxiv.org/abs/1311.4706

  • nice use of dN/dS
  • about 20% of ant gene families show positive selection
  • mostly negative results of what is not under social selection

Araújo, M.B., Ferri-Yáñez, F., Bozinovic, F., Marquet, P.A., Valladares, F. & Chown, S.L. (2013). Heat freezes niche evolution. Ecology Letters, 16, 1206–1219.

  • paper mentioned by Lacy.
  • greater constraints in CTmax than CTmin across species

Signorotti, L., Jaisson, P. & d’ Ettorre, P. (2014). Larval memory affects adult nest-mate recognition in the ant Aphaenogaster senilis. Proceedings of the Royal Society B: Biological Sciences, 281, 20132579.

  • colonies accept adoptive larvae! I knew this from composite colonies…
  • authors also show that once eclosed, larvae are less aggresive to both adoptive and genetically-related workers

Brown, S., Savage, P.E., Ko, A.M.-S., Stoneking, M., Ko, Y.-C., Loo, J.-H. & Trejaut, J.A. (2014). Correlations in the population structure of music, genes and language. Proceedings of the Royal Society B: Biological Sciences, 281, 20132072.

  • title says it: correlations between structure of music and genes
  • authors claim “quantitative evidence that music and genes may have coevolved by demonstrating significant correlations between traditional group-level folk songs and mitochondrial DNA variation”
  • can this be statistical wishfullness? seems to bizarre to be true…
  • correlation between music and genes statistically significant (r=0.42, p=0.015) and remained significant when geographical distance controlled for (r=0.385, p=0.032)
  • MARGINALLY significant after correcting for multiple testing
  • laughable “…music can be shown to be correlated with a robust genetic marker like mtDNA suggests that it might have a sufficient time-depth to track population movements”. even if true, why would you want to???

Pickrell, J. (2013). Joint analysis of functional genomic data and genome-wide association studies of 18 human traits. bioRxiv. Retrieved November 19, 2013, from http://biorxiv.org/content/early/2013/11/19/000752

  • method to utilize functional data to improve GWAS
  • assign prior probablity to SNP based on annotation

Johnson, V.E. (2013). Revised standards for statistical evidence. Proceedings of the National Academy of Sciences, 201313476.

  • another criticism of frequentist methods


climate cascade

In group meeting, discussed R markdown, data formatting and worked on analyzing a qPCR dataset of Andrew’s. Used ddply to efficiently calculate slope for 8 targets!

ddply(datasub, .(target), summarize, slope = lm(Ct ~ log(dilution))$coef[2])


Gradually have realized that having all analyses for ‘climate cascade’ project in a single git repository is neither achievable or practical. Makes much more sense to have a github organization or lab (e.g GotelliLab) that has a fork of each individual’s github repository for a specific project or analysis. In this vein, transferring ApTranscriptome project from within climate-cascade repository to a stand-alone project and repository. At same time, redoing analysis with the following changes:

  • Assembly both Ar and A22 together
  • Run assembly in Trinity only
  • Perform digital normalization included in Trinity
  • include --min_kmer_cov 2 parameter which uses only those kmers occurring at least twice. this will control for Illumina sequencing error-rate

Then, as before, use UCLUST and CAP3 to remove redundant transcripts and merge overlapping transcripts, respectively. Map reads from each sample to assembly individually.


Phone call with Sonia Dobias, Qiagen Technical Service Project Leader

  • Buffer ATL proteinase K lysis buffer
  • Buffer G2 not too different
  • after digest, should be fairly clear, somewhat viscous
  • DNA purified using DNeasy average molecular weight 30-60kb
  • this should be more than adequate
  • DNA purified using Genomic-tip not sheared, much longer 150kb
  • recommended strategy
    • put all ants in mortar, pour liquid nitrogen on and grind into powder with pestle
    • scrape powder into tube and incubate with ATL overnight
    • perform standard extraction from this point!


Guide to using ggplot2

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