Plaque size tool: an easy bioinformatics instrument for phage plaque size measurements

Issue 144 | September 17, 2021
10 min read
Capsid and Tail

Intermediate binary file with phage plaques processed by Plaque Size Tool. Photo credit: Ellina Trofimova.

This week, we’re featuring a post by Ellina Trofimova, software engineer-turned-MSc student at Macquarie University in Australia. She tells us about the Plaque Size Tool she’s developed, including how it came about and what it can do!

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Phage Futures EU 2021 returns in-person in Brussels, Belgium on November 23-24! The theme will be ‘Translating phage-based applications into clinically and commercially viable therapeutics’.

The ‘super earlybird’ deadline is Oct 1. And you can use code PHAGEDIRECTORY10 (or PD10) for a 10% discount!

Download the agenda here.

What’s New

Marion Bichet (Monash University, Australia) and colleagues published a protocol in Star Protocols for studying phage interactions with in vitro epithelial cell layers. They developed a simplified method for high-titer phage purification and cleanup and a comprehensive technique to look at phage-epithelial cell interaction under the microscope. They also describe the use of transwells to study microorganism transcytosis and whole-cell analysis for quantification of intracellular phage particles.

Epithelial cellsProtocol

Graça Pinto (University of Minho, Portugal) and colleagues published a research article in FEMS Microbiology ecology on the interactions of phage ace and shiga toxin-producing Escherichia coli during biocontrol. This work demonstrated that phage Ace is a safe biocontrol agent against STEC contamination and that the emergence of bacteriophage insensitive mutants did not represent a greater risk in environmental persistence and human pathogenicity.

Biocontrol agentProphageToxin

Andrzej Zielezinski (Adam Mickiewicz University, Poland) and colleagues published a new preprint describing PHIST, a fast and accurate prediction of prokaryotic hosts from metagenomic viral sequences.

Bioinformatics ToolHost predictionMetagenomicsPreprint

Ian Rambo (University of Texas Austin) and colleagues published a preprint on unique viruses that infect Archaea related to eukaryotes. Here they characterized seven viral genomes that infected Lokiarchaeota, Helarchaeota, and Thorarchaeota in deep-sea hydrothermal sediments.

Deep sea ventsViral genome

Aisling Brady (University of Glasgow, UK) and colleagues published a review article in Annual Reviews of Microbiology on the molecular basis of lysis–lysogeny decisions in Gram-positive phages. They discuss a variety of mechanisms and the genetic organization of these systems.

LysogenyMolecular mechanisms

Latest Jobs

Gut viromeResearch ScientistVirome
The Quadram Institute Bioscience (QIB), based in Norwich, UK is hiring a Virome Research Scientist to study the intestinal virome and understanding the diversity and impact of viruses at key life stages during human development. The successful applicant will work with a number of phage researchers including the Carding, Adriaenssens and Clokie groups.
PhD projectPhage Therapy
The Iredell lab at the University of Sydney is seeking a PhD student to undertake research in phage therapy for intracellular pathogens.
Gut microbiotaPost DocViromics
The Shkoporov lab at APC Microbiome, Ireland is hiring a Post-Doctoral Researcher to work on a new project, PHAGENET, which involves investigating the role of phages in horizontal gene transfer in the human gut microbiome.
PhD projectPhage-host interactionsPost Doc
The Papenfort laboratory at the Friedrich Schiller University of Jena (Germany) studies gene regulation in microbial pathogens, and is recruiting postdocs and/or PhD students to employ genetic tools and synthetic RNAs to study phage-host interactions and identify and chracterize novel phage resistance mechansims.
Post DocViral ecologyViromics
Friedrich Schiller University Jena is looking for a Postdoctoral Microbiological Laboratory Manager to coordinate and support wet lab activities and pursue own research interests within the scope of Viral Ecology and Omics.
Bioinformatic toolsBioinformatician
Friedrich Schiller University Jena is looking for a bioinformatic programmer/data steward to assist in the establishment and maintenance of a compute cluster, databases, bioinformatics tools, building pipelines, and much more.

Community Board

Anyone can post a message to the phage community — and it could be anything from collaboration requests, post-doc searches, sequencing help — just ask!

Greetings, I hope everyone is doing well during these trying times. My name is Jonathan Lalsiamthara, a Post Doc trainee at the Aballay Lab, OHSU, Portland, OR. I am reaching out to the community if anyone will share with us the cystoviruses or dsRNA bacteriophages (eg. phi6, phi8, phi12, phi13, phi2954, phiNN, and phiYY). I am interested in studying host-pathogen interaction using the C. elegans model, and we assure the requested strains will be used for research purposes only and not to be shared with a third party. I will be very grateful if you will reach me at lalsiamt@ohsu.edu to discuss any further details. Thank you and Best Wishes- Jonathan, Portland

Seeking phage for research

As part of the ‘Meet-a-Scientist’ series, the Science Journal for Kids and Teens interviewed Tiffany Luong, PhD student and a phage scientist at San Diego State University. Check out the interview here to find out more about ‘Can virsues save lives?’.

Celebrating phage scientistsQ&AResources

The Centre of Microbiome Science (Ohio State University) is organizing a Viromics workshop webinar series (virtual) on October 13-15, 2021. The workshop will cover virus identification and classification tools, and virus databases. See the full agenda and register here.

Phage workshopsVirus tools

For PHAVES #22 next week, Dr. Saima Aslam, MD (IPATH, UCSD, California) will present ‘Clinical phage therapy: Learning from patients’ on Sept 20 at 9AM Pacific! Hear about her experience in treating patients with phage and ask her all your questions! Register here!

Virtual EventPHAVESPhage Therapy

African Phage Forum (APF) is hosting the sixth session of their webinar series on September 24 at 7 AM EAT/9 AM PDT. Guest speaker Dr. Ivan Liachko will be presenting his talk on “Culture-Free Assembly and Host-Attribution of Phage Genomes in Metagenomes”. Register for this event or upcoming events here.

Phage genomeToolVirtual Event

Stephanie Lynch will host the next Phage Phun session, which will be Wednesday September 29 at 3PM Pacific (an Australian friendly time!). Join us for this informal virtual networking session by registering here. (If you’re already signed up for the PHAVES series or a past Phage Phun, no need to register again; we’ll send you the link closer to the date).

FunPhage phun!Virtual Event

Plaque size tool: an easy bioinformatics instrument for phage plaque size measurements

Profile Image
MSc Student
Macquarie University
Twitter @ellinium_

A software engineer who switched to science.

About two years ago, I completed an internship at Dr Paul Jaschke’s lab at Macquarie University (Sydney, Australia). I was trying to evolve a decompressed phiX174 bacteriophage created by my supervisor several years ago at Stanford University. One of the tasks was to measure plaques of the evolved phage. I looked online for the tools to ease my work and found a couple that seemed suitable, but I struggled with their installation. After installation, I realised that they required some additional settings that weren’t easy to understand, and they could not process plaques with bull’s eye morphology (plaques with a clear centre and one or several outer turbid concentric rings) either. So, I gave up, found a ruler in a drawer of one of the lab benches and measured the plaques manually. The work was finally done.

This year I needed to measure plaques of different sizes and morphology for my own project; some of the plaques were extremely small, and it would have taken forever to assess them manually. So, this time, I’ve decided to refresh my software engineering skills and created a plaque size measurement tool called Plaque Size Tool based on the OpenCV Python library. It was published in Virology (Elsevier) in 2021, and its files are available on GitHub.

Installation

Because of my previous frustrations with installing various tools, I’ve tried to make the installation as easy as possible. It requires pre-installed Python 3, but all other packages are installed automatically, so there is no need to install them manually one by one. I’ve also added screenshots to the manual so you can follow along.

Validation

To check the tool, I first tested it on 499 plaques formed by different variants of phiX174 phage on E. coli C and found that the results did not significantly differ from the manual measurement in Fiji (where you need to circle each plaque manually). After that, I looked for other images of plates with phage plaques and found an extensive collection in the Actinobacteriophage database. Processed images of various formats and quality can be found in the Supplementary File of our paper.

During peer review of the Plaque Size Tool, one of the reviewer’s questions was whether the tool could handle turbid plaques as they do not have clear edges due to incomplete lysis. With the great help of the Phage Directory community, we were able to obtain additional plate images with turbid plaques and measure their plaque size. Overall, the tool can measure between 70 and 100% plaques on a plate for plaque sizes greater than 1.5 mm and 51-91% for plaques less than 1.5 mm. Although not designed to measure titres, if all plaques are distributed on the plate without forming overlaps, it is possible to get the total number of plaques on a plate. The tool can measure sizes of plaques of quite different morphology: clear, bull’s eye, turbid, and plaques with clear centres and turbid edges.

Additional options

The tool can measure the plaque size (diameter, area) either in pixels or in mm. To get the value in mm, a plate size in mm needs to be specified in the option -p (for example, -p 90 means that the plate on the image is 90 mm in diameter).

The PST can process individual images (-i option) and bulk if a directory with images is specified (-d option). If you’re processing photos with small plaques (less than 1.5 mm), another option could be used: -small. In addition to the output image with valid plaques outlined with green colour and ID with diameter specified for each plaque, a CSV file is also formed containing all plaque IDs with corresponding Area and Diameter values both in pixels and mm if plate size is specified.

The example of the images processed with the PST shows two plates: one with small plaques (top) and another with average plaques (bottom):

Plate example

As testing was done on a large but still limited sample of images, it is possible that the tool might not process particular images very well (for example, the images are too bright or dark or have noise). In that case, I would appreciate it if you could send me an email (ellina.trofimova@hdr.mq.edu.au) or create an issue on GitHub to fix a particular issue and ease your work of phage size measurement. The tool works under all OSes supporting Python 3, is open source (under Apache License 2.0), can be used in any automatic pipelines and modified by users for other purposes.

Many thanks to Atif Khan and Stephanie Lynch for finding, summarizing and editing this week’s phage news, jobs and community posts! And thanks to Lizzie Richardson for editing the feature article!

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