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

PLoS One. 2018 Aug 3;13(8):e0201174. doi: 10.1371/journal.pone.0201174. eCollection 2018.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Biodiversity
  • DNA, Bacterial / genetics
  • DNA, Bacterial / isolation & purification
  • DNA, Fungal / genetics
  • DNA, Fungal / isolation & purification
  • Feces / microbiology
  • Female
  • Gastrointestinal Microbiome / genetics*
  • Humans
  • Male
  • Microbial Consortia / genetics
  • Microbiological Techniques
  • Middle Aged
  • Mycobiome / genetics

Substances

  • DNA, Bacterial
  • DNA, Fungal

Grant support

This study was supported by a grant from Pfizer and the Société Française de Mycologie Médicale (Prix de Recherche SFMM - Pfizer 2015) to CA. MEB received institutional grant funding from MSD and Astellas and lecture fees or travel grants from MSD, Astellas, and Gilead Sciences. FB received grants from Astellas, and payment for lectures from Merck. The specific roles of these authors are articulated in the 'author contributions' section. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.