Active Microbiome Structure and Functional Analyses of Freshwater Benthic Biofilm Samples Influenced by RNA Extraction Methods

Yuan Yao; Subramanya Rao; Olivier Habimana

doi:10.3389/fmicb.2021.588025

Active Microbiome Structure and Functional Analyses of Freshwater Benthic Biofilm Samples Influenced by RNA Extraction Methods

Yuan Yao, Subramanya Rao, Olivier Habimana^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

2 Scopus citations

Abstract

Advances in high-throughput sequencing technologies have enabled extensive studies of freshwater biofilms and significant breakthroughs in biofilm meta-omics. To date, however, no standardized protocols have been developed for the effective isolation of RNA from freshwater benthic biofilms. In this study, we compared column-based kit RNA extraction with five RNAzol-based extractions, differentiated by various protocol modifications. The RNA products were then evaluated to determine their integrity, purity and yield and were subjected to meta-transcriptomic sequencing and analysis. Significant discrepancies in the relative abundance of active communities and structures of eukaryotic, bacterial, archaebacterial, and viral communities were observed as direct outcomes of the tested RNA extraction methods. The column isolation-based group was characterized by the highest relative abundance of Archaea and Eukaryota, while the organic isolation-based groups commonly had the highest relative abundances of Prokaryota (bacteria). Kit extraction methods provided the best outcomes in terms of high-quality RNA yield and integrity. However, these methods were deemed questionable for studies of active bacterial communities and may contribute a significant degree of bias to the interpretation of downstream meta-transcriptomic analyses.

Original language	English
Article number	588025
Journal	Frontiers in Microbiology
Volume	12
DOIs	https://doi.org/10.3389/fmicb.2021.588025
State	Published - 16 Apr 2021
Externally published	Yes

Keywords

16S rRNA gene sequencing
RNA extraction
benthic biofilms
freshwater ecosystems
meta-transcriptomics
method comparison

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.3389/fmicb.2021.588025

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title = "Active Microbiome Structure and Functional Analyses of Freshwater Benthic Biofilm Samples Influenced by RNA Extraction Methods",

abstract = "Advances in high-throughput sequencing technologies have enabled extensive studies of freshwater biofilms and significant breakthroughs in biofilm meta-omics. To date, however, no standardized protocols have been developed for the effective isolation of RNA from freshwater benthic biofilms. In this study, we compared column-based kit RNA extraction with five RNAzol-based extractions, differentiated by various protocol modifications. The RNA products were then evaluated to determine their integrity, purity and yield and were subjected to meta-transcriptomic sequencing and analysis. Significant discrepancies in the relative abundance of active communities and structures of eukaryotic, bacterial, archaebacterial, and viral communities were observed as direct outcomes of the tested RNA extraction methods. The column isolation-based group was characterized by the highest relative abundance of Archaea and Eukaryota, while the organic isolation-based groups commonly had the highest relative abundances of Prokaryota (bacteria). Kit extraction methods provided the best outcomes in terms of high-quality RNA yield and integrity. However, these methods were deemed questionable for studies of active bacterial communities and may contribute a significant degree of bias to the interpretation of downstream meta-transcriptomic analyses.",

keywords = "16S rRNA gene sequencing, RNA extraction, benthic biofilms, freshwater ecosystems, meta-transcriptomics, method comparison",

author = "Yuan Yao and Subramanya Rao and Olivier Habimana",

note = "Publisher Copyright: {\textcopyright} Copyright {\textcopyright} 2021 Yao, Rao and Habimana.",

year = "2021",

month = apr,

day = "16",

doi = "10.3389/fmicb.2021.588025",

language = "英语",

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journal = "Frontiers in Microbiology",

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T1 - Active Microbiome Structure and Functional Analyses of Freshwater Benthic Biofilm Samples Influenced by RNA Extraction Methods

AU - Yao, Yuan

AU - Rao, Subramanya

AU - Habimana, Olivier

PY - 2021/4/16

Y1 - 2021/4/16

N2 - Advances in high-throughput sequencing technologies have enabled extensive studies of freshwater biofilms and significant breakthroughs in biofilm meta-omics. To date, however, no standardized protocols have been developed for the effective isolation of RNA from freshwater benthic biofilms. In this study, we compared column-based kit RNA extraction with five RNAzol-based extractions, differentiated by various protocol modifications. The RNA products were then evaluated to determine their integrity, purity and yield and were subjected to meta-transcriptomic sequencing and analysis. Significant discrepancies in the relative abundance of active communities and structures of eukaryotic, bacterial, archaebacterial, and viral communities were observed as direct outcomes of the tested RNA extraction methods. The column isolation-based group was characterized by the highest relative abundance of Archaea and Eukaryota, while the organic isolation-based groups commonly had the highest relative abundances of Prokaryota (bacteria). Kit extraction methods provided the best outcomes in terms of high-quality RNA yield and integrity. However, these methods were deemed questionable for studies of active bacterial communities and may contribute a significant degree of bias to the interpretation of downstream meta-transcriptomic analyses.

AB - Advances in high-throughput sequencing technologies have enabled extensive studies of freshwater biofilms and significant breakthroughs in biofilm meta-omics. To date, however, no standardized protocols have been developed for the effective isolation of RNA from freshwater benthic biofilms. In this study, we compared column-based kit RNA extraction with five RNAzol-based extractions, differentiated by various protocol modifications. The RNA products were then evaluated to determine their integrity, purity and yield and were subjected to meta-transcriptomic sequencing and analysis. Significant discrepancies in the relative abundance of active communities and structures of eukaryotic, bacterial, archaebacterial, and viral communities were observed as direct outcomes of the tested RNA extraction methods. The column isolation-based group was characterized by the highest relative abundance of Archaea and Eukaryota, while the organic isolation-based groups commonly had the highest relative abundances of Prokaryota (bacteria). Kit extraction methods provided the best outcomes in terms of high-quality RNA yield and integrity. However, these methods were deemed questionable for studies of active bacterial communities and may contribute a significant degree of bias to the interpretation of downstream meta-transcriptomic analyses.

KW - 16S rRNA gene sequencing

KW - RNA extraction

KW - benthic biofilms

KW - freshwater ecosystems

KW - meta-transcriptomics

KW - method comparison

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U2 - 10.3389/fmicb.2021.588025

DO - 10.3389/fmicb.2021.588025

M3 - 文章

AN - SCOPUS:85105175542

SN - 1664-302X

VL - 12

JO - Frontiers in Microbiology

JF - Frontiers in Microbiology

M1 - 588025

ER -

Active Microbiome Structure and Functional Analyses of Freshwater Benthic Biofilm Samples Influenced by RNA Extraction Methods

Abstract

Keywords

UN SDGs

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