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      OpenComet: An automated tool for comet assay image analysis

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          Abstract

          Reactive species such as free radicals are constantly generated in vivo and DNA is the most important target of oxidative stress. Oxidative DNA damage is used as a predictive biomarker to monitor the risk of development of many diseases. The comet assay is widely used for measuring oxidative DNA damage at a single cell level. The analysis of comet assay output images, however, poses considerable challenges. Commercial software is costly and restrictive, while free software generally requires laborious manual tagging of cells. This paper presents OpenComet, an open-source software tool providing automated analysis of comet assay images. It uses a novel and robust method for finding comets based on geometric shape attributes and segmenting the comet heads through image intensity profile analysis. Due to automation, OpenComet is more accurate, less prone to human bias, and faster than manual analysis. A live analysis functionality also allows users to analyze images captured directly from a microscope. We have validated OpenComet on both alkaline and neutral comet assay images as well as sample images from existing software packages. Our results show that OpenComet achieves high accuracy with significantly reduced analysis time.

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          Highlights

          • •

            OpenComet is a free software tool for measuring DNA damage in comet assay images.

          • •

            An automated image analysis algorithm extracts, segments and scores comet shapes.

          • •

            OpenComet is validated to be accurate with respect to manual scoring.

          • •

            Due to automation, OpenComet is less prone to human bias and reduces analysis time.

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          Most cited references24

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          A simple technique for quantitation of low levels of DNA damage in individual cells.

          Joseph McCoy, Raymond R. Tice, V. Singh … (1988)
          Human lymphocytes were either exposed to X-irradiation (25 to 200 rads) or treated with H2O2 (9.1 to 291 microM) at 4 degrees C and the extent of DNA migration was measured using a single-cell microgel electrophoresis technique under alkaline conditions. Both agents induced a significant increase in DNA migration, beginning at the lowest dose evaluated. Migration patterns were relatively homogeneous among cells exposed to X-rays but heterogeneous among cells treated with H2O2. An analysis of repair kinetics following exposure to 200 rads X-rays was conducted with lymphocytes obtained from three individuals. The bulk of the DNA repair occurred within the first 15 min, while all of the repair was essentially complete by 120 min after exposure. However, some cells demonstrated no repair during this incubation period while other cells demonstrated DNA migration patterns indicative of more damage than that induced by the initial irradiation with X-rays. This technique appears to be sensitive and useful for detecting damage and repair in single cells.
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            Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay.

            P Olive, Sarah Durand, J Banáth (1990)
            A method for measuring DNA damage to individual cells, based on the technique of microelectrophoresis, was described by Ostling and Johanson in 1984 (Biochem. Biophys. Res. Commun. 123, 291-298). Cells embedded in agarose are lysed, subjected briefly to an electric field, stained with a fluorescent DNA-binding stain, and viewed using a fluorescence microscope. Broken DNA migrates farther in the electric field, and the cell then resembles a "comet" with a brightly fluorescent head and a tail region which increases as damage increases. We have used video image analysis to define appropriate "features" of the comet as a measure of DNA damage, and have quantified damage and repair by ionizing radiation. The assay was optimized for lysing solution, lysing time, electrophoresis time, and propidium iodide concentration using Chinese hamster V79 cells. To assess heterogeneity of response of normal versus malignant cells, damage to both tumor cells and normal cells within mouse SCC-VII tumors was assessed. Tumor cells were separated from macrophages using a cell-sorting method based on differential binding of FITC-conjugated goat anti-mouse IgG. The "tail moment", the product of the amount of DNA in the tail and the mean distance of migration in the tail, was the most informative feature of the comet image. Tumor and normal cells showed significant heterogeneity in damage produced by ionizing radiation, although the average amount of damage increased linearly with dose (0-15 Gy) and suggested similar net radiosensitivities for the two cell types. Similarly, DNA repair rate was not significantly different for tumor and normal cells, and most of the cells had repaired the damage by 30 min following exposure to 15 Gy. The heterogeneity in response did not appear to be a result of differences in response through the cell cycle.
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              A cross-platform public domain PC image-analysis program for the comet assay.

              Krzysztof Końca, Anna Lankoff, Anna Banasik … (2003)
              The single-cell gel electrophoresis, also known as the comet assay, has gained wide-spread popularity as a simple and reliable method to measure genotoxic and cytotoxic effects of physical and chemical agents as well as kinetics of DNA repair. Cells are generally stained with fluorescent dyes. The analysis of comets--damaged cells which form a typical comet-shaped pattern--is greatly facilitated by the use of a computer image-analysis program. Although several image-analysis programs are available commercially, they are expensive and their source codes are not provided. For Macintosh computers a cost-free public domain macro is available on the Internet. No ready for use, cost-free program exists for the PC platform. We have, therefore, developed such a public domain program under the GNU license for PC computers. The program is called CASP and can be run on a variety of hardware and software platforms. Its practical merit was tested on human lymphocytes exposed to gamma-rays and found to yield reproducible results. The binaries for Windows 95 and Linux, together with the source code can be obtained from: http://www.casp.of.pl.
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                Author and article information

                Contributors
                Benjamin M. Gyori
                Gireedhar Venkatachalam
                P.S. Thiagarajan
                David Hsu
                Marie-Veronique Clement
                Journal
                Journal ID (nlm-ta): Redox Biol
                Journal ID (iso-abbrev): Redox Biol
                Title: Redox Biology
                Publisher: Elsevier
                ISSN (Electronic): 2213-2317
                Publication date PMC-release: 9 January 2014
                Publication date (Electronic): 9 January 2014
                Publication date Collection: 2014
                Volume: 2
                Pages: 457-465
                Affiliations
                [a ]NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore
                [b ]Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
                [c ]School of Computing, National University of Singapore, Singapore
                Author notes
                [* ]Correspondence to: Block MD6, Centre for Translational Medicine, 14 Medical Drive, #14-01T, Singapore 117599, Singapore. Tel.: +65 6516 7985. bchmvc@ 123456nus.edu.sg
                [1]

                Authors contributed equally.

                Article
                Publisher ID: S2213-2317(14)00003-2
                DOI: 10.1016/j.redox.2013.12.020
                PMC ID: 3949099
                PubMed ID: 24624335
                SO-VID: 5de8ee65-ee59-4775-8e27-e38b76f26e46
                Copyright © © 2014 The Authors
                History
                Date received : 6 November 2013
                Date revision received : 18 December 2013
                Date accepted : 22 December 2013
                Categories
                Subject: Method

                Keywords: comet assay,single cell gel electrophoresis,dna damage,image processing,intensity profile analysis,imagej plug-in
                Data availability:
                Keywords: comet assay, single cell gel electrophoresis, dna damage, image processing, intensity profile analysis, imagej plug-in

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