000095382 001__ 95382
000095382 005__ 20210902121825.0
000095382 0247_ $$2doi$$a10.3389/fmicb.2020.01093
000095382 0248_ $$2sideral$$a119191
000095382 037__ $$aART-2020-119191
000095382 041__ $$aeng
000095382 100__ $$aLejal, E.
000095382 245__ $$aTaxon Appearance From Extraction and Amplification Steps Demonstrates the Value of Multiple Controls in Tick Microbiota Analysis
000095382 260__ $$c2020
000095382 5060_ $$aAccess copy available to the general public$$fUnrestricted
000095382 5203_ $$aBackground: The development of high-throughput sequencing technologies has substantially improved analysis of bacterial community diversity, composition, and functions. Over the last decade, high-throughput sequencing has been used extensively to identify the diversity and composition of tick microbial communities. However, a growing number of studies are warning about the impact of contamination brought along the different steps of the analytical process, from DNA extraction to amplification. In low biomass samples, e.g., individual tick samples, these contaminants may represent a large part of the obtained sequences, and thus generate considerable errors in downstream analyses and in the interpretation of results. Most studies of tick microbiota either do not mention the inclusion of controls during the DNA extraction or amplification steps, or consider the lack of an electrophoresis signal as an absence of contamination. In this context, we aimed to assess the proportion of contaminant sequences resulting from these steps. We analyzed the microbiota of individual Ixodes ricinus ticks by including several categories of controls throughout the analytical process: homogenization, DNA extraction, and DNA amplification. Results: Controls yielded a significant number of sequences (1, 126–13, 198 mean sequences, depending on the control category). Some operational taxonomic units (OTUs) detected in these controls belong to genera reported in previous tick microbiota studies. In this study, these OTUs accounted for 50.9% of the total number of sequences in our samples, and were considered contaminants. Contamination levels (i.e., the percentage of sequences belonging to OTUs identified as contaminants) varied with tick instar and sex: 76.3% of nymphs and 75% of males demonstrated contamination over 50%, while most females (65.7%) had rates lower than 20%. Contamination mainly corresponded to OTUs detected in homogenization and extraction reagent controls, highlighting the importance of carefully controlling these steps. Conclusion: Here, we showed that contaminant OTUs from sample laboratory processing steps can represent more than half the total sequence yield in sequencing runs, and lead to unreliable results when characterizing tick microbial communities. We thus strongly advise the routine use of negative controls in tick microbiota studies, and more generally in studies involving low biomass samples.
000095382 540__ $$9info:eu-repo/semantics/openAccess$$aby$$uhttp://creativecommons.org/licenses/by/3.0/es/
000095382 590__ $$a5.64$$b2020
000095382 591__ $$aMICROBIOLOGY$$b28 / 136 = 0.206$$c2020$$dQ1$$eT1
000095382 592__ $$a1.701$$b2020
000095382 593__ $$aMicrobiology (medical)$$c2020$$dQ1
000095382 593__ $$aMicrobiology$$c2020$$dQ1
000095382 655_4 $$ainfo:eu-repo/semantics/article$$vinfo:eu-repo/semantics/publishedVersion
000095382 700__ $$0(orcid)0000-0001-7483-046X$$aEstrada-Peña, A.$$uUniversidad de Zaragoza
000095382 700__ $$aMarsot, M.
000095382 700__ $$aCosson, J.F.
000095382 700__ $$aRué, O.
000095382 700__ $$aMariadassou, M.
000095382 700__ $$aMidoux, C.
000095382 700__ $$aVayssier-Taussat, M.
000095382 700__ $$aPollet, T.
000095382 7102_ $$11009$$2773$$aUniversidad de Zaragoza$$bDpto. Patología Animal$$cÁrea Sanidad Animal
000095382 773__ $$g11 (2020), 1093 [10 pp.]$$pFront. microbiol.$$tFRONTIERS IN MICROBIOLOGY$$x1664-302X
000095382 8564_ $$s1285109$$uhttps://zaguan.unizar.es/record/95382/files/texto_completo.pdf$$yVersión publicada
000095382 8564_ $$s505750$$uhttps://zaguan.unizar.es/record/95382/files/texto_completo.jpg?subformat=icon$$xicon$$yVersión publicada
000095382 909CO $$ooai:zaguan.unizar.es:95382$$particulos$$pdriver
000095382 951__ $$a2021-09-02-10:11:13
000095382 980__ $$aARTICLE