Abstract
Microorganisms are ubiquitous in the biosphere, playing a crucial role in both biogeochemistry of the planet and human health. However, identifying these microorganisms and defining their function are challenging. Widely used approaches in comparative metagenomics, 16S amplicon sequencing and whole genome shotgun sequencing (WGS), have provided access to DNA sequencing analysis to identify microorganisms and evaluate diversity and abundance in various environments. However, advances in parallel high-throughput DNA sequencing in the past decade have introduced major hurdles, namely standardization of methods, data storage, reproducible interoperability of results, and data sharing. The National Ecological Observatory Network (NEON), established by the National Science Foundation, enables all researchers to address queries on a regional to continental scale around a variety of environmental challenges and provide high-quality, integrated, and standardized data from field sites across the U.S. As the amount of metagenomic data continues to grow, standardized procedures that allow results across projects to be assessed and compared is becoming increasingly important in the field of metagenomics. We demonstrate the feasibility of using publicly available NEON soil metagenomic sequencing datasets in combination with open access Metagenomics Rapid Annotation using the Subsystem Technology (MG-RAST) server to illustrate advantages of WGS compared to 16S amplicon sequencing. Four WGS and four 16S amplicon sequence datasets, from surface soil samples prepared by NEON investigators, were selected for comparison, using standardized protocols collected at the same locations in Colorado between April-July 2014. The dominant bacterial phyla detected across samples agreed between sequencing methodologies. However, WGS yielded greater microbial resolution, increased accuracy, and allowed identification of more genera of bacteria, archaea, viruses, and eukaryota, and putative functional genes that would have gone undetected using 16S amplicon sequencing. NEON open data will be useful for future studies characterizing and quantifying complex ecological processes associated with changing aquatic and terrestrial ecosystems.
Highlights
Over the past decade, interest in total microbial community composition and dynamics of complex environments has increased significantly
We demonstrate effective use of publicly available National Ecological Observatory Network (NEON) soil metagenomic sequencing datasets hosted on the open access Metagenomics Rapid Annotation using Subsystem Technology (MG-RAST) server [45] to assess the feasibility of employing openly sourced NEON data
Mgm4778744.3) metagenomic samples were selected after performing searches on the Metagenomics Rapid Annotation using the Subsystem Technology (MG-RAST) server with predefined filtering criteria and identifying samples with the greatest number of sequencing reads collected from the same location
Summary
Interest in total microbial community composition and dynamics of complex environments has increased significantly. This is because the estimated total number of microbial cells in the earth’s biosphere exceeds 1030 [1], and the microbes themselves harbor potentially up to an additional 1031 phages [2]. Microbial communities have been defined using culture dependent methods to detect and enumerate microorganisms. It is estimated that the vast majority of prokaryotic genospecies remain uncultured [11], and genomes of uncultured microorganisms encode a largely untapped reservoir of novel metabolites and metabolic processes [12]. That major accomplishment has allowed in depth comparison and exploration of microbial ecology [14,15], including the metabolic profile of complex microbial ecosystems [16,17]
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