AUTHOR=Morra Melissa , Marradi Denise , Gandini Luca , Ivagnes Vittorio , Ottolini Giulia , Bovio Alessandro , Jabali Grace , Maraschi Lorenzo , Dada Ifeoluwa Ayomide , Chawanda Tonderai Vitalis , Gorla Martina , Tarasiuk Olga , Mocchetti Chiara , Soluri Maria Felicia , Boccafoschi Francesca , Sblattero Daniele , Cotella Diego 

TITLE=A non-hypothesis-driven practical laboratory activity on functional metagenomics: “fishing” protein-coding DNA sequences from microbiomes

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=Volume 13 - 2025

YEAR=2025

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2025.1602982

DOI=10.3389/fbioe.2025.1602982

ISSN=2296-4185

ABSTRACT=Practical laboratory of the most functional metagenomics courses focuses on activities aimed at providing specific skills in bioinformatics through the analysis of genomic datasets. However, sequence-based analyses of metagenomes should be complemented by function-based analyses, to provide evidential knowledge of gene function. A “true” functional metagenomic approach relies on the construction and screening of metagenomic libraries - physical libraries that contain DNA cloned from metagenomes of various origin. The information obtained from functional metagenomics will help in future annotations of gene function and serve as a complement to sequence-based metagenomics. Here, we describe a simple protocol for the construction of a metagenomic DNA library, optimized and tested by a team of undergraduate biotechnology students. This protocol is based on a technique developed in our laboratory and currently used for research. Using this protocol, libraries of protein domains can be quickly generated, from the DNA of any intron-less genome, such as those of bacteria or phages. Therefore, these libraries provide a valuable platform for training students in various validation tools, including computational methods - for example, metagenome assembly, functional annotation - and proteomics techniques, including protein expression and analysis. By varying the biological source and validation pipeline, this approach offers virtually limitless opportunities for innovative thesis research projects.