PHENOS: a high-throughput and flexible tool for microorganism growth phenotyping on solid media

Abstract

BackgroundMicrobial arrays, with a large number of different strains on a single plate printed with robotic precision, underpin an increasing number of genetic and genomic approaches. These include Synthetic Genetic Array analysis, high-throughput Quantitative Trait Loci (QTL) analysis and 2-hybrid techniques. Measuring the growth of individual colonies within these arrays is an essential part of many of these techniques but is useful for any work with arrays. Measurement is typically done using intermittent imagery fed into complex image analysis software, which is not especially accurate and is challenging to use effectively. We have developed a simple and fast alternative technique that uses a pinning robot and a commonplace microplate reader to continuously measure the thickness of colonies growing on solid agar, complemented by a technique for normalizing the amount of cells initially printed to each spot of the array in the first place. We have developed software to automate the process of combining multiple sets of readings, subtracting agar absorbance, and visualizing colony thickness changes in a number of informative ways.ResultsThe “PHENOS” pipeline (PHENotyping On Solid media), optimized for Saccharomyces yeasts, produces highly reproducible growth curves and is particularly sensitive to low-level growth. We have empirically determined a formula to estimate colony cell count from an absorbance measurement, and shown this to be comparable with estimates from measurements in liquid. We have also validated the technique by reproducing the results of an earlier QTL study done with conventional liquid phenotyping, and found PHENOS to be considerably more sensitive.Conclusions“PHENOS” is a cost effective and reliable high-throughput technique for quantifying growth of yeast arrays, and is likely to be equally very useful for a range of other types of microbial arrays. A detailed guide to the pipeline and software is provided with the installation files at https://github.com/gact/phenos.Electronic supplementary materialThe online version of this article (10.1186/s12866-017-1143-y) contains supplementary material, which is available to authorized users.

Restoring fertility in yeast hybrids: Breeding and quantitative genetics of beneficial traits

“…Fitness analysis was done following two different strategies, using either Phenosuite software (Singer Instruments) or the PHENOS platform ( 94 ). Details of the phenotypic analysis are given in SI Appendix , Supplementary Methods .…”

Functional and transcriptional profiling of non-coding RNAs in yeast reveal context-dependent phenotypes and in trans effects on the protein regulatory network

“…For example, CUT494/SUT053/SUT468Δ, SUT129Δ, SUT329Δ and CUT442Δ exhibited fitness impairment in YP + 2% glycerol which was not previously detected in the solid fitness assay. Discrepancies between solid and liquid fitness are likely due to the differing oxygen availability and diffusion rates of one or more nutrients on solid media [30][31][32][33][34][35]. Indeed, when growing on solid surfaces, colony morphology differs between yeast growth phases and time [35][36][37].…”

Restoring fertility in yeast hybrids: breeding and quantitative genetics of beneficial traits

“…The colony sizes were calculated in pixels using Phenosuite software (Singer Instruments, UK) and the heat maps of the phenotypic behaviours were constructed using the R shiny app (https://kobchai-180 shinyapps01.shinyapps.io/heatmap_construction/). In strategy two, phenotyping was performed using the PHENOS platform (Barton et al 2018). Change in absorbance (600 nm) was measured using the FLUOstar Omega plate reader (BMG Labtech).…”