Impact of Preservation Method and 16S rRNA Hypervariable Region on Gut Microbiota Profiling

ABSTRACT Proper preservation of stool samples to minimize microbial community shifts and inactivate infectious agents is important for self-collected specimens requiring shipment to laboratories when cold chain transport is not feasible. In this study, we evaluated the performance of six preservatio...

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Autores principales:	Zigui Chen, Pak Chun Hui, Mamie Hui, Yun Kit Yeoh, Po Yee Wong, Martin C. W. Chan, Martin C. S. Wong, Siew C. Ng, Francis K. L. Chan, Paul K. S. Chan
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	16S rRNA gene amplicon sequencing bacterial culture gut microbiota preservative Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/feeb04fa8b7746eb87f71139dee72b4c
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spelling	oai:doaj.org-article:feeb04fa8b7746eb87f71139dee72b4c2021-12-02T19:47:33ZImpact of Preservation Method and 16S rRNA Hypervariable Region on Gut Microbiota Profiling10.1128/mSystems.00271-182379-5077https://doaj.org/article/feeb04fa8b7746eb87f71139dee72b4c2019-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00271-18https://doaj.org/toc/2379-5077ABSTRACT Proper preservation of stool samples to minimize microbial community shifts and inactivate infectious agents is important for self-collected specimens requiring shipment to laboratories when cold chain transport is not feasible. In this study, we evaluated the performance of six preservation solutions (Norgen, OMNI, RNAlater, CURNA, HEMA, and Shield) for these aspects. Following storage of human stool samples with these preservatives at room temperature for 7 days, three hypervariable regions of the bacterial 16S rRNA gene (V1-V2, V3-V4, and V4) were amplicon sequenced. We found that samples collected in two preservatives, Norgen and OMNI, showed the least shift in community composition relative to −80°C standards compared with other storage conditions, and both efficiently inhibited the growth of aerobic and anaerobic bacteria. RNAlater did not prevent bacterial activity and exhibited relatively larger community shift. Although the effect of preservation solution was small compared to intersubject variation, notable changes in microbiota composition were observed, which could create biases in downstream data analysis. When community profiles inferred from different 16S rRNA gene hypervariable regions were compared, we found differential sensitivity of primer sets in identifying overall microbial community and certain bacterial taxa. For example, reads generated by the V4 primer pair showed a higher alpha diversity of the gut microbial community. The degenerate 27f-YM primer failed to detect the majority of Bifidobacteriales. Our data indicate that choice of preservation solution and 16S rRNA gene primer pair are critical determinants affecting gut microbiota profiling. IMPORTANCE Large-scale human microbiota studies require specimens collected from multiple sites and/or time points to maximize detection of the small effects in microbe-host interactions. However, batch biases caused by experimental protocols, such as sample collection, massively parallel sequencing, and bioinformatics analyses, remain critical and should be minimized. This work evaluated the effects of preservation solutions and bacterial 16S rRNA gene primer pairs in revealing human gut microbiota composition. Since notable changes in detecting bacterial composition and abundance were observed among choice of preservatives and primer pairs, a consistent methodology is essential in minimizing their effects to facilitate comparisons between data sets.Zigui ChenPak Chun HuiMamie HuiYun Kit YeohPo Yee WongMartin C. W. ChanMartin C. S. WongSiew C. NgFrancis K. L. ChanPaul K. S. ChanAmerican Society for Microbiologyarticle16S rRNA geneamplicon sequencingbacterial culturegut microbiotapreservativeMicrobiologyQR1-502ENmSystems, Vol 4, Iss 1 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	16S rRNA gene amplicon sequencing bacterial culture gut microbiota preservative Microbiology QR1-502
spellingShingle	16S rRNA gene amplicon sequencing bacterial culture gut microbiota preservative Microbiology QR1-502 Zigui Chen Pak Chun Hui Mamie Hui Yun Kit Yeoh Po Yee Wong Martin C. W. Chan Martin C. S. Wong Siew C. Ng Francis K. L. Chan Paul K. S. Chan Impact of Preservation Method and 16S rRNA Hypervariable Region on Gut Microbiota Profiling
description	ABSTRACT Proper preservation of stool samples to minimize microbial community shifts and inactivate infectious agents is important for self-collected specimens requiring shipment to laboratories when cold chain transport is not feasible. In this study, we evaluated the performance of six preservation solutions (Norgen, OMNI, RNAlater, CURNA, HEMA, and Shield) for these aspects. Following storage of human stool samples with these preservatives at room temperature for 7 days, three hypervariable regions of the bacterial 16S rRNA gene (V1-V2, V3-V4, and V4) were amplicon sequenced. We found that samples collected in two preservatives, Norgen and OMNI, showed the least shift in community composition relative to −80°C standards compared with other storage conditions, and both efficiently inhibited the growth of aerobic and anaerobic bacteria. RNAlater did not prevent bacterial activity and exhibited relatively larger community shift. Although the effect of preservation solution was small compared to intersubject variation, notable changes in microbiota composition were observed, which could create biases in downstream data analysis. When community profiles inferred from different 16S rRNA gene hypervariable regions were compared, we found differential sensitivity of primer sets in identifying overall microbial community and certain bacterial taxa. For example, reads generated by the V4 primer pair showed a higher alpha diversity of the gut microbial community. The degenerate 27f-YM primer failed to detect the majority of Bifidobacteriales. Our data indicate that choice of preservation solution and 16S rRNA gene primer pair are critical determinants affecting gut microbiota profiling. IMPORTANCE Large-scale human microbiota studies require specimens collected from multiple sites and/or time points to maximize detection of the small effects in microbe-host interactions. However, batch biases caused by experimental protocols, such as sample collection, massively parallel sequencing, and bioinformatics analyses, remain critical and should be minimized. This work evaluated the effects of preservation solutions and bacterial 16S rRNA gene primer pairs in revealing human gut microbiota composition. Since notable changes in detecting bacterial composition and abundance were observed among choice of preservatives and primer pairs, a consistent methodology is essential in minimizing their effects to facilitate comparisons between data sets.
format	article
author	Zigui Chen Pak Chun Hui Mamie Hui Yun Kit Yeoh Po Yee Wong Martin C. W. Chan Martin C. S. Wong Siew C. Ng Francis K. L. Chan Paul K. S. Chan
author_facet	Zigui Chen Pak Chun Hui Mamie Hui Yun Kit Yeoh Po Yee Wong Martin C. W. Chan Martin C. S. Wong Siew C. Ng Francis K. L. Chan Paul K. S. Chan
author_sort	Zigui Chen
title	Impact of Preservation Method and 16S rRNA Hypervariable Region on Gut Microbiota Profiling
title_short	Impact of Preservation Method and 16S rRNA Hypervariable Region on Gut Microbiota Profiling
title_full	Impact of Preservation Method and 16S rRNA Hypervariable Region on Gut Microbiota Profiling
title_fullStr	Impact of Preservation Method and 16S rRNA Hypervariable Region on Gut Microbiota Profiling
title_full_unstemmed	Impact of Preservation Method and 16S rRNA Hypervariable Region on Gut Microbiota Profiling
title_sort	impact of preservation method and 16s rrna hypervariable region on gut microbiota profiling
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/feeb04fa8b7746eb87f71139dee72b4c
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Impact of Preservation Method and 16S rRNA Hypervariable Region on Gut Microbiota Profiling

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