Refinement of 16S rRNA gene analysis for low biomass biospecimens

Abstract High-throughput phylogenetic 16S rRNA gene analysis has permitted to thoroughly delve into microbial community complexity and to understand host-microbiota interactions in health and disease. The analysis comprises sample collection and storage, genomic DNA extraction, 16S rRNA gene amplifi...

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Main Authors: Remy Villette, Gaelle Autaa, Sophie Hind, Johanna B. Holm, Alicia Moreno-Sabater, Martin Larsen
Format: Article
Language:English
Published: Nature Publishing Group 2021-05-01
Series:Scientific Reports
Online Access:https://doi.org/10.1038/s41598-021-90226-2
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spelling doaj-1818d4b5788f4d449b131b0ed8fa2f392021-05-30T11:37:07ZengNature Publishing GroupScientific Reports2045-23222021-05-0111111210.1038/s41598-021-90226-2Refinement of 16S rRNA gene analysis for low biomass biospecimensRemy Villette0Gaelle Autaa1Sophie Hind2Johanna B. Holm3Alicia Moreno-Sabater4Martin Larsen5Inserm UMR-S1135, Centre d’Immunologie et des Maladies Infectieuses (CIMI-Paris)Inserm UMR-S1135, Centre d’Immunologie et des Maladies Infectieuses (CIMI-Paris)Sorbonne Universités, UPMC Univ Paris 06, CR7, Centre d’Immunologie et des Maladies Infectieuses (CIMI-Paris), Hôpital Pitié-SalpêtrièreDepartment of Microbiology and Immunology, Institute for Genome Sciences, University of Maryland School of MedicineInserm UMR-S1135, Centre d’Immunologie et des Maladies Infectieuses (CIMI-Paris)Inserm UMR-S1135, Centre d’Immunologie et des Maladies Infectieuses (CIMI-Paris)Abstract High-throughput phylogenetic 16S rRNA gene analysis has permitted to thoroughly delve into microbial community complexity and to understand host-microbiota interactions in health and disease. The analysis comprises sample collection and storage, genomic DNA extraction, 16S rRNA gene amplification, high-throughput amplicon sequencing and bioinformatic analysis. Low biomass microbiota samples (e.g. biopsies, tissue swabs and lavages) are receiving increasing attention, but optimal standardization for analysis of low biomass samples has yet to be developed. Here we tested the lower bacterial concentration required to perform 16S rRNA gene analysis using three different DNA extraction protocols, three different mechanical lysing series and two different PCR protocols. A mock microbiota community standard and low biomass samples (108, 107, 106, 105 and 104 microbes) from two healthy donor stools were employed to assess optimal sample processing for 16S rRNA gene analysis using paired-end Illumina MiSeq technology. Three DNA extraction protocols tested in our study performed similar with regards to representing microbiota composition, but extraction yield was better for silica columns compared to bead absorption and chemical precipitation. Furthermore, increasing mechanical lysing time and repetition did ameliorate the representation of bacterial composition. The most influential factor enabling appropriate representation of microbiota composition remains sample biomass. Indeed, bacterial densities below 106 cells resulted in loss of sample identity based on cluster analysis for all tested protocols. Finally, we excluded DNA extraction bias using a genomic DNA standard, which revealed that a semi-nested PCR protocol represented microbiota composition better than classical PCR. Based on our results, starting material concentration is an important limiting factor, highlighting the need to adapt protocols for dealing with low biomass samples. Our study suggests that the use of prolonged mechanical lysing, silica membrane DNA isolation and a semi-nested PCR protocol improve the analysis of low biomass samples. Using the improved protocol we report a lower limit of 106 bacteria per sample for robust and reproducible microbiota analysis.https://doi.org/10.1038/s41598-021-90226-2
collection DOAJ
language English
format Article
sources DOAJ
author Remy Villette
Gaelle Autaa
Sophie Hind
Johanna B. Holm
Alicia Moreno-Sabater
Martin Larsen
spellingShingle Remy Villette
Gaelle Autaa
Sophie Hind
Johanna B. Holm
Alicia Moreno-Sabater
Martin Larsen
Refinement of 16S rRNA gene analysis for low biomass biospecimens
Scientific Reports
author_facet Remy Villette
Gaelle Autaa
Sophie Hind
Johanna B. Holm
Alicia Moreno-Sabater
Martin Larsen
author_sort Remy Villette
title Refinement of 16S rRNA gene analysis for low biomass biospecimens
title_short Refinement of 16S rRNA gene analysis for low biomass biospecimens
title_full Refinement of 16S rRNA gene analysis for low biomass biospecimens
title_fullStr Refinement of 16S rRNA gene analysis for low biomass biospecimens
title_full_unstemmed Refinement of 16S rRNA gene analysis for low biomass biospecimens
title_sort refinement of 16s rrna gene analysis for low biomass biospecimens
publisher Nature Publishing Group
series Scientific Reports
issn 2045-2322
publishDate 2021-05-01
description Abstract High-throughput phylogenetic 16S rRNA gene analysis has permitted to thoroughly delve into microbial community complexity and to understand host-microbiota interactions in health and disease. The analysis comprises sample collection and storage, genomic DNA extraction, 16S rRNA gene amplification, high-throughput amplicon sequencing and bioinformatic analysis. Low biomass microbiota samples (e.g. biopsies, tissue swabs and lavages) are receiving increasing attention, but optimal standardization for analysis of low biomass samples has yet to be developed. Here we tested the lower bacterial concentration required to perform 16S rRNA gene analysis using three different DNA extraction protocols, three different mechanical lysing series and two different PCR protocols. A mock microbiota community standard and low biomass samples (108, 107, 106, 105 and 104 microbes) from two healthy donor stools were employed to assess optimal sample processing for 16S rRNA gene analysis using paired-end Illumina MiSeq technology. Three DNA extraction protocols tested in our study performed similar with regards to representing microbiota composition, but extraction yield was better for silica columns compared to bead absorption and chemical precipitation. Furthermore, increasing mechanical lysing time and repetition did ameliorate the representation of bacterial composition. The most influential factor enabling appropriate representation of microbiota composition remains sample biomass. Indeed, bacterial densities below 106 cells resulted in loss of sample identity based on cluster analysis for all tested protocols. Finally, we excluded DNA extraction bias using a genomic DNA standard, which revealed that a semi-nested PCR protocol represented microbiota composition better than classical PCR. Based on our results, starting material concentration is an important limiting factor, highlighting the need to adapt protocols for dealing with low biomass samples. Our study suggests that the use of prolonged mechanical lysing, silica membrane DNA isolation and a semi-nested PCR protocol improve the analysis of low biomass samples. Using the improved protocol we report a lower limit of 106 bacteria per sample for robust and reproducible microbiota analysis.
url https://doi.org/10.1038/s41598-021-90226-2
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