Taxon Appearance From Extraction and Amplification Steps Demonstrates the Value of Multiple Controls in Tick Microbiota Analysis
BackgroundThe 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 microbia...
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Frontiers Media S.A.
2020-06-01
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Online Access: | https://www.frontiersin.org/article/10.3389/fmicb.2020.01093/full |
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doaj-05d9915e6fd84839a3f7dfb0dd4c524e |
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record_format |
Article |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Emilie Lejal Agustín Estrada-Peña Maud Marsot Jean-François Cosson Olivier Rué Olivier Rué Mahendra Mariadassou Mahendra Mariadassou Cédric Midoux Cédric Midoux Cédric Midoux Muriel Vayssier-Taussat Thomas Pollet Thomas Pollet |
spellingShingle |
Emilie Lejal Agustín Estrada-Peña Maud Marsot Jean-François Cosson Olivier Rué Olivier Rué Mahendra Mariadassou Mahendra Mariadassou Cédric Midoux Cédric Midoux Cédric Midoux Muriel Vayssier-Taussat Thomas Pollet Thomas Pollet Taxon Appearance From Extraction and Amplification Steps Demonstrates the Value of Multiple Controls in Tick Microbiota Analysis Frontiers in Microbiology contaminant low biomass sample tick microbiota high-throughput sequencing |
author_facet |
Emilie Lejal Agustín Estrada-Peña Maud Marsot Jean-François Cosson Olivier Rué Olivier Rué Mahendra Mariadassou Mahendra Mariadassou Cédric Midoux Cédric Midoux Cédric Midoux Muriel Vayssier-Taussat Thomas Pollet Thomas Pollet |
author_sort |
Emilie Lejal |
title |
Taxon Appearance From Extraction and Amplification Steps Demonstrates the Value of Multiple Controls in Tick Microbiota Analysis |
title_short |
Taxon Appearance From Extraction and Amplification Steps Demonstrates the Value of Multiple Controls in Tick Microbiota Analysis |
title_full |
Taxon Appearance From Extraction and Amplification Steps Demonstrates the Value of Multiple Controls in Tick Microbiota Analysis |
title_fullStr |
Taxon Appearance From Extraction and Amplification Steps Demonstrates the Value of Multiple Controls in Tick Microbiota Analysis |
title_full_unstemmed |
Taxon Appearance From Extraction and Amplification Steps Demonstrates the Value of Multiple Controls in Tick Microbiota Analysis |
title_sort |
taxon appearance from extraction and amplification steps demonstrates the value of multiple controls in tick microbiota analysis |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Microbiology |
issn |
1664-302X |
publishDate |
2020-06-01 |
description |
BackgroundThe 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.ResultsControls 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.ConclusionHere, 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. |
topic |
contaminant low biomass sample tick microbiota high-throughput sequencing |
url |
https://www.frontiersin.org/article/10.3389/fmicb.2020.01093/full |
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doaj-05d9915e6fd84839a3f7dfb0dd4c524e2020-11-25T03:03:26ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2020-06-011110.3389/fmicb.2020.01093492638Taxon Appearance From Extraction and Amplification Steps Demonstrates the Value of Multiple Controls in Tick Microbiota AnalysisEmilie Lejal0Agustín Estrada-Peña1Maud Marsot2Jean-François Cosson3Olivier Rué4Olivier Rué5Mahendra Mariadassou6Mahendra Mariadassou7Cédric Midoux8Cédric Midoux9Cédric Midoux10Muriel Vayssier-Taussat11Thomas Pollet12Thomas Pollet13UMR BIPAR, Animal Health Laboratory, INRAE, ANSES, Ecole Nationale Vétérinaire d’Alfort, Université Paris-Est, Maisons-Alfort, FranceFaculty of Veterinary Medicine, University of Zaragoza, Zaragoza, SpainLaboratory for Animal Health, Epidemiology Unit, ANSES, University Paris-Est, Maisons-Alfort, FranceUMR BIPAR, Animal Health Laboratory, INRAE, ANSES, Ecole Nationale Vétérinaire d’Alfort, Université Paris-Est, Maisons-Alfort, FranceINRAE, MaIAGE, Université Paris-Saclay, Jouy-en-Josas, FranceINRAE, Bioinfomics, MIGALE Bbioinformatics Facility, Université Paris-Saclay, Jouy-en-Josas, FranceINRAE, MaIAGE, Université Paris-Saclay, Jouy-en-Josas, FranceINRAE, Bioinfomics, MIGALE Bbioinformatics Facility, Université Paris-Saclay, Jouy-en-Josas, FranceINRAE, MaIAGE, Université Paris-Saclay, Jouy-en-Josas, FranceINRAE, Bioinfomics, MIGALE Bbioinformatics Facility, Université Paris-Saclay, Jouy-en-Josas, FranceINRAE, PROSE, Université Paris-Saclay, Antony, FranceAnimal Health Department, INRAE, Nouzilly, FranceUMR BIPAR, Animal Health Laboratory, INRAE, ANSES, Ecole Nationale Vétérinaire d’Alfort, Université Paris-Est, Maisons-Alfort, FranceUMR ASTRE, CIRAD, INRAE, Montpellier, FranceBackgroundThe 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.ResultsControls 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.ConclusionHere, 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.https://www.frontiersin.org/article/10.3389/fmicb.2020.01093/fullcontaminantlow biomass sampletickmicrobiotahigh-throughput sequencing |