Mar 27, 2026
Version 2
Refining Feulgen: low-cost and accurate genome size measurements for everyone V.2
- Mohammed M.Tawfeeq1,2,
- Ursula Swaelus3,
- Florence Rodriguez Gaudray3,
- Jenny Arrensdorff3,
- Felipe Ennes Silva1,4,
- Laurent Grumiau3,
- Thomas Verdebout5,
- Jean-François Flot1,6,7
- 1Department of Organismal Biology, Université libre de Bruxelles (ULB), Av. F.D. Roosevelt, CP 160/12, Brussels, 1050, Belgium;
- 2Interuniversity Institute of Bioinformatics in Brussels – (IB)², Belgium.;
- 3Department of Organismal Biology, Université libre de Bruxelles (ULB), Av. F.D. Roosevelt, CP 160/12, Brussels, 1050, Belgium.;
- 4Department of Anthropology, New York University, USA.;
- 5Department of Mathematics, Université libre de Bruxelles (ULB), Campus La Plaine, Bd du Triomphe, CP 210, Bruxelles, 1050, Belgium.;
- 6Interuniversity Institute of Bioinformatics in Brussels – (IB)², Belgium;
- 7Brussels Laboratory of the Universe - BLU, Belgium.
External link: https://www.biorxiv.org/content/10.1101/2025.08.29.673164v2
Document Citation: Mohammed M.Tawfeeq, Ursula Swaelus, Florence Rodriguez Gaudray, Jenny Arrensdorff, Felipe Ennes Silva, Laurent Grumiau, Thomas Verdebout, Jean-François Flot 2026. Refining Feulgen: low-cost and accurate genome size measurements for everyone. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm3by9l3p/v2Version created by Mohammed M.TAWFEEQ
Manuscript citation:
Mohammed M. Tawfeeq, Ursula Swaelus, Florence Rodriguez Gaudray, Jenny Arrensdorff, Felipe Ennes Silva, Laurent Grumiau, Thomas Verdebout, Jean-François Flot
bioRxiv 2025.08.29.673164; doi: https://doi.org/10.1101/2025.08.29.673164
License: This is an open access document distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: March 24, 2026
Last Modified: March 27, 2026
Document Integer ID: 313836
Keywords: Feulgen Image Analysis Densitometry, C-value, DNA content, genome size, accurate genome size measurement, biodiversity genomic, huge advantage for biodiversity genomic, first time the genome size, using whole genome sequencing, whole genome sequencing, everyone feulgen image analysis densitometry, refinements on the feulgen protocol, black garden ant lasius niger as standard, feulgen protocol, refining feulgen, other methods such as flow cytometry, red bald uakari monkey cacajao rubicundus, flow cytometry, specimen, american cockroach periplaneta americana, fresh tissue sample
Funders Acknowledgements:
Belgian Fonds de la Recherche Scientifique (F.R.S.-FNRS), "Projet de Recherches"
Grant ID: T.0078.23
”Action de Recherche Concertée” to Jean-François Flot and via ‘Chargé de Recherches'
Grant ID: 40017464
Abstract
Feulgen image analysis densitometry (FIAD) has been extensively used for decades to measure genome sizes, but is usually considered less precise and less reproducible compared to other methods such as flow cytometry (FCM). However, FCM requires fresh tissue samples, which is a major impediment to using it on specimens collected in the field far from the lab. By contrast, FIAD works on ethanol-preserved samples kept at room temperature, which is a huge advantage for biodiversity genomics. In this study, we present refinements on the Feulgen protocol and downstream analyses that produce measurements at least as reliable and precise as those obtained using whole genome sequencing. We illustrate it by measuring for the first time the genome size of the red bald uakari monkey Cacajao rubicundus using the American cockroach Periplaneta americana and the black garden ant Lasius niger as standards.
For detailed information, check our preprint on this link :
For detailed protocol, check out the supplementary files on this link :
Acknowledgements
This study was supported by the Belgian Fonds de la Recherche Scientifique (F.R.S.-FNRS) via ‘Projet de Recherches’ grant T.0078.23 and “Action de Recherche Concertée” to JFF and via ‘Chargé de Recherches’ grant 40017464 to FES. We thank Clément Thijs and Stéphane Cherrier for providing the insects used as standards, and Olivier De Thier for useful discussions.