Seasonal dynamics in the mammalian microbiome between disparate environments

# Seasonal dynamics in the mammalian microbiome between disparate environments This README was generated on 2023-11-03 by Mason Stothart GENERAL INFORMATION 1. Title of Dataset: Grey squirrel feces 16S rRNA gene amplicon Meta-Data and R script 2. Author Information A. Principal Investigator Contact Information Name: Amy E.M. Newman Institution: University of Guelph Address: 474-570 Gordon St, Guelph, ON N1G 1Y2 Email: [[e-mail]](mailto:[e-mail]) B. Associate or Co-investigator Contact Information Name: Mason Stothart Institution: University of Calgary Address: 3280 Hospital Drive NW Calgary Email: [[e-mail]](mailto:[e-mail]) 3. Date of data collection (single date, range, approximate date): 2016-05-01 - 2018-03-01 4. Geographic location of data collection : Guelph Ontario, Canada & rare Charitable Research Reserve Cambridge, Canada 5. Information about funding sources that supported the collection of the data: This work was funded through Natural Sciences and Engineering Research Council of Canada (NSERC) and University of Guelph funds to A.E.M., a American Society of Mammalogists grant-in-aid of research to M.R.S., a rare Charitable Research Reserve ‘Ages Foundation Fellowship’ to M.R.S.; M.R.S. was supported by an Ontario Graduate Scholarship and NSERC CGS-M while completing this research. SHARING/ACCESS INFORMATION 1. Licenses/restrictions placed on the data: N/A 2. Links to publications that cite or use the data: N/A 3. Links to other publicly accessible locations of the data: N/A 4. Links/relationships to ancillary data sets: 5. Was data derived from another source? yes/no A. If yes, list source(s): No 6. Recommended citation for this dataset: TBD DATA & FILE OVERVIEW File List: * grey-squirrel_microbiome_metadata_2023-09-25.csv * grey-squirrel_16S-OTU-tree.phylip.tre * EcoEvo_Stothart-et-al_2023-09-25.R * Eco-Evo_Grey-Squirrel_Seasonal-Microbiome.biom Relationship between files, if important: Files correspond to sample meta-data, phylogenetic tree constructed from 16S rRNA gene amplicon sequencing data, R script used for data analysis, and raw 16S rRNA gene amplicon .biom table containing operational taxonomic unit (OTU) taxonomy and counts across samples. Additional related data collected that was not included in the current data package: N/A Are there multiple versions of the dataset? yes/no A. If yes, name of file(s) that was updated: No METHODOLOGICAL INFORMATION Description of methods used for collection/generation of data: N/A Methods for processing the data: 16S rRNA gene amplicon sequencing and bioinformatics We extracted DNA from 0.2 g of feces using QIAamp DNA Stool Mini Kits (Qiagen, Hilden Germany). The v4 region of the 16S rRNA gene was amplified in triplicate (primers, 515F: 5’-GTGYCAGCMGCCGCGGTAA-3’; 806R: 5’-GGACTACNVGGGTWTCTAAT-3’) at MetaGenomBio Inc. (Waterloo, Canada; Walters et al., 2015). Triplicate PCR products were pooled and sequenced to a depth of 30,000 reads/sample on an Illumina MiSeq platform (v2 chemistry). Briefly, we merged paired-end reads and performed quality filtering using a standardized mothur pipeline (Kozich et al., 2013). Paired-end reads were clustered to operational taxonomic units (OTUs) based on a 97% 16S rRNA gene sequence similarity using the OptiClust algorithm (Westcott & Schloss, 2017). Taxonomy was assigned to representative v4 sequences using the SILVA v138 reference database (Yilmaz et al., 2014). Only OTUs which were present at a relative abundance of 0.001 in at least a single sample were retained for analysis to remove singletons and potential sequencing errors. OTU counts were rarefied to the lowest read-count in the dataset (3326 reads) prior to all analyses. References Kozich, J. J., Westcott, S. L., Baxter, N. T., Highlander, S. K., & Schloss, P. D. (2013). Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the miseq illumina sequencing platform. *Applied and Environmental Microbiology*, *79*(17), 5112–5120. [https://doi.org/10.1128/AEM.01043-13](https://doi.org/10.1128/AEM.01043-13) Walters, W., Hyde, E. R., Berg-lyons, D., Ackermann, G., Humphrey, G., Parada, A., Gilbert, J. a, & Jansson, J. K. (2015). Improved bacterial 16S rRNA gene (V4 and V4-5) and fungal internal transcribed spacer marker gene primers for microbial community surveys. *mSystems*, *1*(1), 1–10. [https://doi.org/10.1128/mSystems.00009-15.Editor](https://doi.org/10.1128/mSystems.00009-15.Editor) Westcott, S. L., & Schloss, P. D. (2017). OptiClust, an improved method for assigning amplicon-based sequence data to operational taxonomic units. *mSphere*, *2*(2), 1–11. [https://doi.org/10.1128/mspheredirect.00073-17](https://doi.org/10.1128/mspheredirect.00073-17) Yilmaz, P., Parfrey, L. W., Yarza, P., Gerken, J., Pruesse, E., Quast, C., Schweer, T., Peplies, J., Ludwig, W., & Glöckner, F. O. (2014). The SILVA and ‘‘All-species Living Tree Project (LTP)’’ taxonomic frameworks. Nucleic Acid Research, 42, 643–648. [https://doi.org/10.1093/nar/gkt1209](https://doi.org/10.1093/nar/gkt1209) Instrument- or software-specific information needed to interpret the data: R v4.2.1, R packages: ape (v-5.6-2), phyloseq (v-1.40.0, genefilter (v-1.78.0), vegan (v-2.6-2), microbiome (v-1.18.0), ANCOMBC (v-1.6.2), ALDEx2 (v-1.26.0), breakaway (v-4.8.4) Standards and calibration information, if appropriate: N/A Environmental/experimental conditions: Wild Describe any quality-assurance procedures performed on the data: Validated against sequencing negative controls People involved with sample collection, processing, analysis and/or submission: Mason Stothart, Hayley Spina, Michelle Hotchkiss, Winnie Ko, and Amy Newman DATA-SPECIFIC INFORMATION FOR: grey-squirrel_microbiome_metadata_2023-09-25.csv 1. Number of variables: 12 2. Number of cases/rows: 118 3. Variable List: | Variable | Description | | ----------------------- | --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | sample\_names | unique ID corresponding to fecal samples and sequence fasta files. | | year | year of sample collection | | month | month of sample collection | | day | day of month of sample collection | | season | Season of sample collection (Fall, Winter, Spring, Summer) | | fecal\_ID | Sample ID with year indicator | | left\_tag | squirrel left ear tag alphanumeric code | | right\_tag | squirrel right ear tag alphanumeric code | | environment | environment in which focal animal was captured | | mass | mass of squirrel in grams | | sex | sex of squirrel (male = M, female = F) | | reproductive\_condition | reproductive condition of squirrel at time of capture (R = female in estrus, NR = female in non-reproductive condition, SCR = male with external testes, NSCR = male with no external testes) | EcoEvo_Stothart-et-al_2023-09-25.R Description: An R file containing scripts used to process and analyze data contained within the Grey-squirrel_microbiome_metadata_2023-09-25.csv, grey-squirrel_16S-OTU-tree.phylip.tre, and Eco-Evo_Grey-Squirrel_Seasonal-Microbiome.biom data files. This includes commands for importing microbiome data into a 'phyloseq' format, alpha diversity analyses using the 'breakaway' package, tests of beta diversity using PERMANOVA tests, and differential abundance tests using the packages ANCOMBC and ALDEx2. R version 4.2.1 Loaded R packages: ape (v-5.6-2), phyloseq (v-1.40.0, genefilter (v-1.78.0), vegan (v-2.6-2), microbiome (v-1.18.0), ANCOMBC (v-1.6.2), ALDEx2 (v-1.26.0), breakaway (v-4.8.4) Missing data codes: N/A Specialized formats or other abbreviations used: N/A