The National Ecological Observatory Network (NEON) annually performs shotgun metagenomic sequencing to sample genes within soils at 47 sites across the United States. NEON serves as a valuable educational resource, thanks to its open data policies and programming tutorials, but there is currently no introductory tutorial for performing analyses with the soil shotgun metagenomic dataset. Here, we describe a workflow for processing raw soil metagenome sequencing reads using the Sunbeam bioinformatics pipeline. The workflow includes cleaning and processing raw reads, taxonomic classification, assembly into contigs, annotation of predicted genes using custom protein databases, and exporting assemblies to the KBase platform for downstream analysis. This workflow is designed to be robust to annual data releases from NEON, and the underlying Snakemake framework can manage complex software dependencies. The workflow presented here aims to increase the accessibility of NEON’s shotgun metagenome data, which can provide important clues about soil microbial communities and their ecological roles.

The largest dataset of soil metagenomes has recently been released by the National Ecological Observatory Network (NEON), which performs annual shotgun sequencing of soils at 47 sites across the United States. NEON serves as a valuable educational resource, thanks to its open data and programming tutorials, but there is currently no introductory tutorial for accessing and analyzing the soil shotgun metagenomic dataset. Here, we describe methods for processing raw soil metagenome sequencing reads using a bioinformatics pipeline tailored to the high complexity and diversity of the soil microbiome. We describe the rationale, necessary resources, and implementation of steps such as cleaning raw reads, taxonomic classification, assembly into contigs or genomes, annotation of predicted genes using custom protein databases, and exporting data for downstream analysis. The workflow presented here aims to increase the accessibility of NEON’s shotgun metagenome data, which can provide important clues about soil microbial communities and their ecological roles.