Optimised biomolecular extraction for metagenomic analysis of microbial biofilms from high-mountain streams

PeerJ

article

Susheel Bhanu Busi¹  Paraskevi Pramateftaki²  Jade Brandani²  Stilianos Fodelianakis²  Hannes Peter²  Rashi Halder¹  Paul Wilmes¹  Tom J. Battin² 

[1] Systems Ecology Research Group, Luxembourg Centre for Systems Biomedicine, University of Luxembourg;Stream Biofilm and Ecosystems Research group

关键词: Biofilms;    Streams;    Alpine streams;    Glacier fed streams;    Glaciers;    Metagenomics;    Biomolecular extraction;   

DOI  :  10.7717/peerj.9973

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

Glacier-fed streams (GFS) are harsh ecosystems dominated by microbial life organized in benthic biofilms, yet the biodiversity and ecosystem functions provided by these communities remain under-appreciated. To better understand the microbial processes and communities contributing to GFS ecosystems, it is necessary to leverage high throughput sequencing. Low biomass and high inorganic particle load in GFS sediment samples may affect nucleic acid extraction efficiency using extraction methods tailored to other extreme environments such as deep-sea sediments. Here, we benchmarked the utility and efficacy of four extraction protocols, including an up-scaled phenol-chloroform protocol. We found that established protocols for comparable sample types consistently failed to yield sufficient high-quality DNA, delineating the extreme character of GFS. The methods differed in the success of downstream applications such as library preparation and sequencing. An adapted phenol-chloroform-based extraction method resulted in higher yields and better recovered the expected taxonomic profile and abundance of reconstructed genomes when compared to commercially-available methods. Affordable and straight-forward, this method consistently recapitulated the abundance and genomes of a mock community, including eukaryotes. Moreover, by increasing the amount of input sediment, the protocol is readily adjustable to the microbial load of the processed samples without compromising protocol efficiency. Our study provides a first systematic and extensive analysis of the different options for extraction of nucleic acids from glacier-fed streams for high-throughput sequencing applications, which may be applied to other extreme environments.

【 授权许可】

CC BY