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Combined bacterial and fungal intestinal microbiota analyses: Impact of storage conditions and DNA extraction protocols.

ABSTRACT: BACKGROUND:The human intestinal microbiota contains a vast community of microorganisms increasingly studied using high-throughput DNA sequencing. Standardized protocols for storage and DNA extraction from fecal samples have been established mostly for bacterial microbiota analysis. Here, we investigated the impact of storage and DNA extraction on bacterial and fungal community structures detected concomitantly. METHODS:Fecal samples from healthy adults were stored at -80°C as such or diluted in RNAlater® and subjected to 2 extraction protocols with mechanical lysis: the Powersoil® MoBio kit or the International Human Microbiota Standard (IHMS) Protocol Q. Libraries of the 12 samples targeting the V3-V4 16S and the ITS1 regions were prepared using Metabiote® (Genoscreen) and sequenced on GS-FLX-454. Sequencing data were analysed using SHAMAN (http://shaman.pasteur.fr/). The bacterial and fungal microbiota were compared in terms of diversity and relative abundance. RESULTS:We obtained 171869 and 199089 quality-controlled reads for 16S and ITS, respectively. All 16S reads were assigned to 41 bacterial genera; only 52% of ITS reads were assigned to 40 fungal genera/section. Rarefaction curves were satisfactory in 3/3 and 2/3 subjects for 16S and ITS, respectively. PCoA showed important inter-individual variability of intestinal microbiota largely overweighing the effect of storage or extraction. Storage in RNAlater® impacted (downward trend) the relative abundances of 7/41 bacterial and 6/40 fungal taxa, while extraction impacted randomly 18/41 bacterial taxa and 1/40 fungal taxon. CONCLUSION:Our results showed that RNAlater® moderately impacts bacterial or fungal community structures, while extraction significantly influences the bacterial composition. For combined bacterial and fungal intestinal microbiota analysis, immediate sample freezing should be preferred when feasible, but storage in RNAlater® remains an option under unfavourable conditions or for concomitant metatranscriptomic analysis; and extraction should rely on protocols validated for bacterial analysis, such as IHMS Protocol Q, and including a powerful mechanical lysis, essential for fungal extraction.

SUBMITTER: Angebault C

PROVIDER: S-EPMC6075747 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Combined bacterial and fungal intestinal microbiota analyses: Impact of storage conditions and DNA extraction protocols.

Angebault Cécile C Ghozlane Amine A Volant Stevenn S Botterel Françoise F d'Enfert Christophe C Bougnoux Marie-Elisabeth ME

PloS one 20180803 8

<h4>Background</h4>The human intestinal microbiota contains a vast community of microorganisms increasingly studied using high-throughput DNA sequencing. Standardized protocols for storage and DNA extraction from fecal samples have been established mostly for bacterial microbiota analysis. Here, we investigated the impact of storage and DNA extraction on bacterial and fungal community structures detected concomitantly.<h4>Methods</h4>Fecal samples from healthy adults were stored at -80°C as such ...[more]

PMID: 30074988

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Project description:BACKGROUND:High-throughput sequencing techniques are used to analyse the diversity of the respiratory microbiota in health and disease. Although extensive data are available regarding bacterial respiratory microbiota, its fungal component remains poorly studied. This is partly due to the technical issues associated with fungal metagenomics analyses. In this study, we compared two DNA extraction protocols and two fungal amplification targets for combined bacterial and fungal targeted amplicon sequencing analyses of the respiratory microbiota. METHODS:Six sputa, randomly selected from routine samples in Mondor Hospital (Creteil, France) and treated anonymously, were tested after bacterial and fungal routine culture. Two of which were spiked with Aspergillus Fumigati and Aspergillus Nigri (105 conidia/mL). After mechanical lysis, DNA was extracted using automated QIAsymphony® extraction (AQE) or manual PowerSoil® MoBio extraction (MPE). DNA yield and purity were compared. DNA extracted from spiked sputa was subjected to (i) real-time PCR for Aspergillus DNA detection and (ii) combined metagenomic analyses targeting barcoded primers for fungal ITS1 and ITS2, and bacterial V1-V2 and V3-V4 16S regions. Amplicon libraries were prepared using MiSeq Reagent V3 kit on Illumina platform. Data were analysed using PyroMIC© and SHAMAN software, and compared with culture results. RESULTS:AQE extraction provided a higher yield of DNA (AQE/MPE DNA ratio = 4.5 [1.3-11]) in a shorter time. The yield of Aspergillus DNA detected by qPCR was similar for spiked sputa regardless of extraction protocol. The extraction moderately impacted the diversity or relative abundances of bacterial communities using targeted amplicon sequencing (2/43 taxa impacted). For fungi, the relative abundances of 4/11 major taxa were impacted and AQE results were closer to culture results. The V1-V2 or V3-V4 and ITS1 or ITS2 targets assessed similarly the diversity of bacterial and fungal major taxa, but ITS2 and V3-V4 detected more minor taxa. CONCLUSION:Our results showed the importance of DNA extraction for combined bacterial and fungal targeted metagenomics of respiratory samples. The extraction protocol can affect DNA yield and the relative abundances of few bacterial but more fungal taxa. For fungal analysis, ITS2 allowed the detection of a greater number of minor taxa compared with ITS1.

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Combined bacterial and fungal intestinal microbiota analyses: Impact of storage conditions and DNA extraction protocols.

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Combined bacterial and fungal intestinal microbiota analyses: Impact of storage conditions and DNA extraction protocols.

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