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dc.contributor.authorKumar, Aditi
dc.contributor.authorGravdal, Kristin
dc.contributor.authorSegal, Jonathan P
dc.contributor.authorSteed, Helen
dc.contributor.authorBrookes, Matthew
dc.contributor.authorAl-Hassi, Hafid Omar
dc.date.accessioned2022-07-04T13:12:09Z
dc.date.available2022-07-04T13:12:09Z
dc.date.issued2022-06-15
dc.identifier.citationKumar A, Gravdal K, Segal JP, Steed H, Brookes MJ, Al-Hassi HO. (2022) Variability in the Pre-Analytical Stages Influences Microbiome Laboratory Analyses. Genes, 13(6):1069. https://doi.org/10.3390/genes13061069en
dc.identifier.issn2073-4425en
dc.identifier.pmid35741831 (pubmed)
dc.identifier.doi10.3390/genes13061069en
dc.identifier.urihttp://hdl.handle.net/2436/624824
dc.description© 2022 The Authors. Published by MDPI. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.3390/genes13061069en
dc.description.abstractIntroduction: There are numerous confounding variables in the pre-analytical steps in the analysis of gut microbial composition that affect data consistency and reproducibility. This study compared two DNA extraction methods from the same faecal samples to analyse differences in microbial composition. Methods: DNA was extracted from 20 faecal samples using either (A) chemical/enzymatic heat lysis (lysis buffer, proteinase K, 95 °C + 70 °C) or (B) mechanical and chemical/enzymatic heat lysis (bead-beating, lysis buffer, proteinase K, 65 °C). Gut microbiota was mapped through the 16S rRNA gene (V3–V9) using a set of pre-selected DNA probes targeting >300 bacteria on different taxonomic levels. Apart from the pre-analytical DNA extraction technique, all other parameters including microbial analysis remained the same. Bacterial abundance and deviations in the microbiome were compared between the two methods. Results: Significant variation in bacterial abundance was seen between the different DNA extraction techniques, with a higher yield of species noted in the combined mechanical and heat lysis technique (B). The five predominant bacteria seen in both (A) and (B) were Bacteroidota spp. and Prevotella spp. (p = NS), followed by Bacillota (p = 0.005), Lachhnospiraceae (p = 0.0001), Veillonella spp. (p < 0.0001) and Clostridioides (p < 0.0001). Conclusion: As microbial testing becomes more easily and commercially accessible, a unified international consensus for optimal sampling and DNA isolation procedures must be implemented for robustness and reproducibility of the results.en
dc.description.sponsorshipThis study was supported by the Research Investment Fund-4 (RIF4) from the University of Wolverhampton, Wolverhampton WV1 1LY UK.en
dc.formatapplication/pdfen
dc.languageeng
dc.language.isoenen
dc.publisherMDPIen
dc.relation.urlhttps://www.mdpi.com/2073-4425/13/6/1069en
dc.rightsLicence for published version: Creative Commons Attribution 4.0 International
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.subjectmicrobiomeen
dc.subjectpre-analysisen
dc.subjectDNAen
dc.subject16S rRNAen
dc.subject.meshBacteria
dc.subject.meshEndopeptidase K
dc.subject.meshDNA
dc.subject.meshDNA, Bacterial
dc.subject.meshRNA, Ribosomal, 16S
dc.subject.meshReproducibility of Results
dc.subject.meshMicrobiota
dc.titleVariability in the pre-analytical stages influences microbiome laboratory analysesen
dc.typeJournal articleen
dc.identifier.eissn2073-4425
dc.identifier.journalGenesen
dc.date.updated2022-07-03T20:22:43Z
dc.contributor.institutionDepartment of Gastroenterology, The Royal Wolverhampton NHS Trust, Wolverhampton WV10 0QP, UK.
pubs.place-of-publicationSwitzerland
dc.date.accepted2022-06-14
rioxxterms.funderUniversity of Wolverhamptonen
rioxxterms.identifier.projectUOW04072022HSen
rioxxterms.versionVoRen
rioxxterms.licenseref.urihttp://creativecommons.org/licenses/by/4.0/en
rioxxterms.licenseref.startdate2022-07-04en
dc.source.volume13
dc.source.issue6
dc.source.beginpage1069
dc.description.versionPublished version
refterms.dateFCD2022-07-04T13:12:01Z
refterms.versionFCDVoR
refterms.dateFOA2022-07-04T13:12:10Z


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Licence for published version: Creative Commons Attribution 4.0 International
Except where otherwise noted, this item's license is described as Licence for published version: Creative Commons Attribution 4.0 International