DOI: 10.5061/dryad.jdfn2z3dv
title: Shallow shotgun sequencing of the microbiome recapitulates 16S amplicon results and provides functional insights

description: Prevailing 16S rRNA gene-amplicon methods for characterizing the bacterial
 microbiome of wildlife are economical, but result in coarse taxonomic
 classifications, are subject to primer and 16S copy number biases, and do
 not allow for direct estimation of microbiome functional potential. While
 deep shotgun metagenomic sequencing can overcome many of these
 limitations, it is prohibitively expensive for large sample sets. We
 evaluated the ability of shallow shotgun metagenomic sequencing to
 characterize taxonomic and functional patterns in the fecal microbiome of
 a model population of feral horses (Sable Island, Canada). Since 2007,
 this unmanaged population has been the subject of an individual-based,
 long-term ecological study. Using deep shotgun metagenomic sequencing, we
 determined the sequencing depth required to accurately characterize the
 horse microbiome. In comparing conventional versus high-throughput shotgun
 metagenomic library preparation techniques, we validate the use of more
 cost-effective lab methods. Finally, we characterize similarities between
 16S amplicon and shallow shotgun characterization of the microbiome and
 demonstrate that the latter recapitulates biological patterns first
 described in a published amplicon dataset. Unlike amplicon data, we
 further demonstrate how shallow shotgun metagenomic data provide useful
 insights about microbiome functional potential which support previously
 hypothesized diet effects in this study system.
description: 16S amplicon, deep shotgun metagenomic, and shallow shotgun
 metagenomic seqeunce characterization of the bacterial microbiome of feral
 horse fecal samples. Fresh, individual-linked fecal samples were collected
 from Sable Island feral horses (Sable Island, Nova Scotia) in 2014, during
 annual surveys of the horse population. Samples were kept on ice in the
 field and stored at -20C on Sable Island for two months, before being
 placed into longterm storage at -80C on the mainland.
description: # Shallow shotgun sequencing of the microbiome recapitulates 16S amplicon
 results and provides functional insights
 [https://doi.org/10.5061/dryad.jdfn2z3dv](https://doi.org/10.5061/dryad.jdfn2z3dv) ## Description of the data and file structure Data and R scripts needed to replicate results in 10.22541/au.164943445.54693239/v1, a methodological comparison of amplicon, Nextera prepared shotgun metagenomic, and iGenomx prepared shotgun metagenomic characterization of the Sable Island feral horse fecal microbiome. ### Files and variables #### File: nexterra-and-IgenomxReSeq\_count-table.csv **Description:** Sequence counts of microbial species by sample, for Nextera XT prepared library, and iGenomx Riptide prepared libraries of the same DNA extract. ##### #### File: iGen-NewExtract\_tax-table.csv **Description:** Taxonomic structure of microbial species from iGenomx prepared libraries of the new DNA extracts. Assignments made using Kaiju and the NCBI nr + euk database. ##### #### File: nexterra-and-IgenomxReSeq\_tax-table.csv **Description:** Taxonomic structure of microbial species from Nextera prepared libraries, and iGenomx prepared libraries of the same DNA extracts. Assignments made using Kaiju and the NCBI nr + euk database. ##### #### File: amplicon\_count-table.csv **Description:** Sequence counts of 16S (v3-v4) amplicon sequence variants by sample. #### File: amplicon\_tax-table.csv **Description:** Taxonomic structure of 16S (v3-v4) amplicon sequence variants. ##### #### File: iGen-NewExtract\_count-table.csv **Description:** Sequence counts of microbial species by sample, for newly extracted samples sequenced on an Illumina NovaSeq using an iGenomx Riptide prepared library. ##### #### File: Molecular-Ecology-Resources\_MetaData.csv **Description:** Meta-data corresponding to all samples. Meta-data descriptions provided below: ##### Variables * Data_ID: Unique ID corresponding to sequence dataset. * sample_ID: ID corresponding to the raw biological sample (fecal sample). * type: Sequencing method used to characterize the fecal microbiome. Either amplicon (16S v3-v4 region) or shotgun metagenomic (Nextera XT or iGenomx Riptide library preparations). * age: Horse age in years. * Parental_status: Parental status of the horse (with or without a foal). * date: Date day-month-year. * day_of_year: Days since Jan 1st of the same year. * latitude_decimal_degrees: Latitude in decimal degrees. * longitude_decimal_degrees: Longitude in decimal degrees. * sandwort_area_meters_squared: area of sandwort (meters squared) contained within 150-m radius spatial buffer centre on point of sample collection. #### File: README\_file.txt **Description:** Text document containing descriptions of project files. #### File: MolEcolResources\_2022-09-13.R **Description:** R scripts needed to replicate analyses in paper. ## Access information Other publicly accessible locations of the data: * NCBI SRA BioProject: #PRJNA880353 ## Version Changes 2024-11-11: Uploaded R scripts "MolEcolResources_2022-09-13.R" missing from initial submission.