Abstract
Deep investigation of the microbiome of food-production and food-processing environments through whole-metagenome sequencing (WMS) can provide detailed information on the taxonomic composition and functional potential of the microbial communities that inhabit them, with huge potential benefits for environmental monitoring programs. However, certain technical challenges jeopardize the application of WMS technologies with this aim, with the most relevant one being the recovery of a sufficient amount of DNA from the frequently low-biomass samples collected from the equipment, tools and surfaces of food-processing plants. Here, we present the first complete workflow, with optimized DNA-purification methodology, to obtain high-quality WMS sequencing results from samples taken from food-production and food-processing environments and reconstruct metagenome assembled genomes (MAGs). The protocol can yield DNA loads >10 ng in >98% of samples and >500 ng in 57.1% of samples and allows the collection of, on average, 12.2 MAGs per sample (with up to 62 MAGs in a single sample) in ~1 week, including both laboratory and computational work. This markedly improves on results previously obtained in studies performing WMS of processing environments and using other protocols not specifically developed to sequence these types of sample, in which <2 MAGs per sample were obtained. The full protocol has been developed and applied in the framework of the European Union project MASTER (Microbiome applications for sustainable food systems through technologies and enterprise) in 114 food-processing facilities from different production sectors.
Key points
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This protocol outlines a procedure for sampling the microbiomes of environments with low-biomass yields such as those in a clean food-processing facility and analyzing them through WMS.
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The procedure includes an optimized DNA-extraction stage to maximize DNA yield and allow WMS-based analysis, offering a more complete analysis of the microbiome than targeted methods currently used in industry and avoiding issues of bias associated with targeted high-throughput sequencing.
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Data availability
Raw reads are available on the Sequence Read Archive of the NCBI under the BioProjects numbers PRJNA897099 (for vegetable facilities), PRJNA941197 (for an ice-cream facility), PRJNA997800 (for meat facilities), PRJNA997821 (for cheese facilities, except those located in Ireland) and PRJNA996188 for control samples. Raw reads for fish-processing factories and Irish cheese factories are available on the European Nucleotide Archive database under the accession numbers PRJEB62794 and PRJEB63604, respectively.
Code availability
The code used for raw reads filtering, assembly and binning into MAGs is available at https://github.com/SegataLab/MASTER-WP5-pipelines.
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Acknowledgements
This work was funded by the European Commission under the European Union’s Horizon 2020 research and innovation program under grant agreement no. 818368 (MASTER). C.B. is grateful to Junta de Castilla y León and the European Social Fund for awarding her a pre-doctoral grant (BOCYL-D-07072020-6). A.P. is grateful to Ministerio de Ciencia e Innovación for awarding her a pre-doctoral grant (PRE2021-098910). N.M.Q. is currently funded by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 101034371. We thank AV Star Systems for their role in creating the Supplementary Video, and M. Coakley and S. Mortensen for their help in its preparation.
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Contributions
M.L., M.P., D.O’N., V.T.M., M.W., A.M., N.S., P.D.C., D.E. and A.A.-O. conceived the study and obtained the funding. J.F.C.-D., C.B., F.D.F., V.V., R.C.R., I.C.-T., C.S., S.D., P.R.-M., N.M.Q., M.D., S.S., S.K. and A.P. performed the samplings at food-processing facilities. D.O’N. and L.M.d.S. designed and tested the improvements in the DNA-extraction protocol, and C.B., F.D.F., V.V., R.C.R. and A.P. tested the different versions of the DNA-extraction protocol for optimization. C.B., F.D.F., R.C.R., I.C.T., C.S., S.D., P.R.-M., N.M.Q., M.D., S.S., S.K. and A.P. applied the improved DNA-extraction protocol on samples from the food industry. F.A., F.P. and N.S. sequenced the extracted DNA. N.C., A.B.-M. and F.P. performed the bioinformatic analyses. J.F.C.-D., F.D.F., V.V., R.C.R., N.C., C.S. and N.M.Q. collated all the information. L.M.d.S., J.F.C.D. and C.B. prepared the figures. J.F.C.D., C.B. and A.A.-O. wrote the manuscript with input from all the authors. All authors read and approved the final manuscript.
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D.O’N. and L.M.d.S. are employees of QIAGEN GmbH. All other authors declare no competing interests.
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Nature Protocols thanks Lena Florl, Andrea Moreno Switt, Bernard Taminiau and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key reference using this protocol
Valentino, V. et al. Food Res. Int. 162, 112202 (2022): https://doi.org/10.1016/j.foodres.2022.112202
Supplementary information
Supplementary Information
Supplementary Fig. 1, Note and Methods
Supplementary Video 1
Microbiome mapping in the food industry: a detailed visual procedure on how to prepare the materials and take the samples at a food-processing facility. The steps that should be followed in the laboratory for sample pre-processing are shown.
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Barcenilla, C., Cobo-Díaz, J.F., De Filippis, F. et al. Improved sampling and DNA extraction procedures for microbiome analysis in food-processing environments. Nat Protoc (2024). https://doi.org/10.1038/s41596-023-00949-x
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DOI: https://doi.org/10.1038/s41596-023-00949-x
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