Biology subjects

Flynn, P.

Publications and source records attributed to Flynn, P..

4 recordsLinked to original sources

bioRxiv2023

Quantifying yeast microtubules and spindles using the Toolkit for Automated Microtubule Tracking (TAMiT)

Fluorescently labeled proteins absorb and emit light, appearing as Gaussian spots in fluorescence imaging. When fluorescent tags are added to cytoskeletal polymers such as microtubules, a line of fluorescence and even non-linear structures results. While much progress has been made in techniques for imaging and microscopy, image analysis is less well developed. Current analysis of fluorescent microtubules uses either manual tools, such as kymographs, or automated software. As a result, our ability to quantify microtubule dynamics and organization from light microscopy remains limited. Despite development of automated microtubule analysis tools for in vitro studies, analysis of images from cells often depends heavily on manual analysis. One of the main reasons for this disparity is the low signal-to-noise ratio in cells, where background fluorescence is typically higher than in reconstituted systems. Here, we present the Toolkit for Automated Microtubule Tracking (TAMiT), which automatically detects, optimizes and tracks fluorescent microtubules in living yeast cells with sub-pixel accuracy. Using basic information about microtubule organization, TAMiT detects linear and curved polymers using a geometrical scanning technique. Images are fit via an optimization problem for the microtubule image parameters that is solved using non-linear least squares in Matlab. We benchmark our software using simulated images and show that it reliably detects microtubules, even at low signal-to-noise ratios. Then, we use TAMiT to measure monopolar spindle microtubule bundle number, length, and lifetime in a large dataset that includes several S. pombe mutants that affect microtubule dynamics and bundling. The results from the automated analysis are consistent with previous work, and suggest a direct role for CLASP/Cls1 in bundling spindle microtubules. We also illustrate automated tracking of single curved astral microtubules in S. cerevisiae, with measurement of dynamic instability parameters. The results obtained with our fully-automated software are similar to results using hand-tracked measurements. Therefore, TAMiT can facilitate automated analysis of spindle and microtubule dynamics in yeast cells.

cell biology↗

bioRxiv2021

Comparative analysis of single nucleotide polymorphisms and microsatellite markers for parentage verification and discovery within the equine Thoroughbred breed

Short tandem repeat (STR), also known as microsatellite markers are currently used for genetic parentage verification within equine. Transitioning from STR to single nucleotide polymorphism (SNP) markers to perform equine parentage verification is now a potentially feasible prospect and a key area requiring evaluation is parentage testing accuracies when using SNP based methods, in comparison to STRs. To investigate, we utilised a targeted equine genotyping by sequencing (GBS) panel of 562 SNPs to SNP genotype 309 Thoroughbred horses - inclusive of 55 previously parentage verified offspring. Availability of STR profiles for all 309 horses, enabled comparison of parentage accuracies between SNP and STR panels. An average sample call rate of 97.2% was initially observed, and subsequent removal of underperforming SNPs realised a pruned final panel of 516 SNPs. Simulated trio and partial parentage scenarios were tested across 12-STR, 16-STR, 147-SNP and 516-SNP panels. False-positives (i.e. expected to fail parentage, but pass) ranged from 0% for 147-SNP and 516-SNP panels to 0.003% when using 12-STRs within trio parentage scenarios, and 0% for 516-SNPs to 1.6% for 12-STRs within partial parentage scenarios. Our study leverages targeted GBS methods to generate low-density equine SNP profiles and demonstrates the value of SNP based equine parentage analysis in comparison to STRs - particularly when performing partial parentage discovery.

genetics↗

bioRxiv2021

A high-content platform for physiological profiling and unbiased classification of individual neurons

High-throughput physiological assays often lose single cell resolution, precluding subtype-specific analyses of neuronal activation mechanism and drug effects. Here, we demonstrate APPOINT, Automated Physiological Phenotyping Of Individual Neuronal Types. This physiological assay platform combines calcium imaging, robotic liquid handling, and automated analysis to generate physiological activation profiles of single neurons at a large scale. Using unbiased techniques, we quantify responses to multiple sequential stimuli, enabling subgroup identification by physiology and probing of distinct mechanisms of neuronal activation within subgroups. Using APPOINT, we quantify primary sensory neuron activation by metabotropic receptor agonists and identify potential contributors to pain signaling. Furthermore, we expand the role of neuroimmune interactions by showing that human serum can directly activate sensory neurons, elucidating a new potential pain mechanism. Finally, we apply APPOINT to develop a high-throughput, all-optical approach for quantification of activation threshold and pharmacologically separate the contributions of distinct ion channel subsets to optical activation.

neuroscience↗

bioRxiv2017

SNP Data Quality Control in a National Beef and Dairy Cattle System and Highly Accurate SNP Based Parentage Verification and Identification

A major use of genetic data is parentage verification and identification as inaccurate pedigrees negatively affect genetic gain. Since 2012 the international standard for single nucleotide polymorphism (SNP) based verification in Bos taurus cattle has been the ISAG 100 and 200 SNP panels. While these SNP sets have provided an increased level of parentage accuracy over microsatellite markers (MS), they can validate the wrong parent for an animal at [≤]1% misconcordance rate levels, indicating that more SNP are needed if a more accurate pedigree is required. With rapidly increasing numbers of cattle being genotyped in Ireland that represent 61 Bos taurus breeds from a wide range of farm types: beef/dairy, AI/pedigree/commercial, purebred/crossbred, and large to small herd size the Irish Cattle Breeding Federation (ICBF) analysed different SNP densities to determine that at a minimum [≥]500 SNP are needed to consistently predict only one set of parents at a [≤]1% misconcordance rate. For parentage validation and prediction ICBF uses 800 SNP selected based on SNP clustering quality, ISAG200 inclusion, call rate (CR), and minor allele frequency (MAF) in the Irish cattle population. Large datasets require sample and SNP quality control (QC). Most publications only deal with SNP QC via CR, MAF, parent-progeny conflicts, and Hardy-Weinberg deviation, but not sample QC. We report here a genomic sample QC pipeline to deal with the unique challenges of >1,000,000 genotypes from a national herd such as SNP genotype errors from mis-tagging of animals, lab errors, farm errors, and multiple other issues that can arise.

genetics↗