Gut microbiomes of mammals and birds from Slovenia: 16S rRNA sequencing data
Description
A large dataset of fecal microbiomes from 715 individual animals representing over 50 mammalian and avian species from Slovenia. We collected samples from both wild and domestic animals with an emphasis on capturing microbial diversity across a wide range of taxa and ecological contexts. The samples were subjected to 16S rRNA gene sequencing, targeting the V3–V4 hypervariable region. Bioinformatic analysis was performed using Usearch to generate zero-radius operational taxonomic units (ZOTUs). This dataset was generated primarily for the development of microbial source tracking (MST) assays used for identifying the sources of fecal pollution in contaminated water. However, it provides a valuable resource for broader microbiome research. It enables comparative studies across host species, trophic guilds, and environmental contexts such as domestication. The Mendeley Data repository contains both raw and processed versions of the ZOTU table, ZOTU sequences in FASTA format, taxonomy table and sample metadata. The raw data is located in the "Raw" folder, while the processed data (filtered to remove non-bacterial sequences and rarefied) can be found in the "Filtered and rarefied" folder. All scripts used for data processing, analysis, and figure generation are provided in the “Code” folder.
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Sampling Fecal samples wild and domestic mammals and birds were collected across Slovenia between December 2020 and September 2023. In total, we obtained 515 mammalian and 200 avian fecal samples. The samples were collected using sterile stool containers with a plastic spoon and immediately stored in ice-filled coolers. Where possible, only the interior of the fecal deposit was sampled to minimize contamination from external sources such as soil, litter and urine. The samples were transported to the laboratory within 24 hours. When immediate transport was not possible, they were stored at -20 °C and later transported to the laboratory on ice. The samples were stored at -80 °C until further processing. DNA isolation and amplicon sequencing The DNA was isolated from 0.25 g of each sample using the QIAamp Fast Stool Mini Kit (Qiagen, Hilden, Germany) with mechanical disruption (MagNA Lyser; 7000 rpm for 70s). The concentrations of DNA were measured using the Quant-iT PicoGreen dsDNA Kit (Thermo Fisher Scientific). The DNA was stored at -80 °C until further processing. The V3-V4 hypervariable region of the 16S rRNA was amplified with the primers Bakt_341F (5′-CCTACGGGNGGCWGCAG-3′) and Bakt_805R (5′-GACTACHVGGGTATCTAATCC-3′). Library preparation followed the Illumina 16S Metagenomic Sequencing Library Preparation protocol (Illumina, CA, USA) and was performed using either KAPA HiFi HotStart ReadyMix (Kapa Biosystems, MA, USA) or Q5 High-Fidelity DNA Polymerase (New England Biolabs, USA). Sequencing was carried out on Illumina platforms using 600-cycle reagent kits. Details on polymerase type and sequencing platform for each sample are provided in the metadata_raw.xlsx file. A 10% PhiX control was included in each run for quality control. Raw sequence analysis Raw sequences were processed using Usearch version 11.0.667. Paired-end reads were merged with the maximum of 15 mismatches in the alignment. Primer sequences were removed and sequences exceeding one expected error were discarded. Unique sequences underwent denoising with the UNOISE algorithm, generating zero-radius operational taxonomic units (ZOTUs). Sequences shorter than 400 bp were excluded, and taxonomy was assigned using the RDP training set (v.19). Filtering and normalization Further filtering was performed using R version 4.2.3. Taxa with confidence scores below 0.8 were categorized as unclassified. Non-bacterial ZOTUs, and sequences classified as chloroplasts at the class level were excluded from the dataset. The ZOTU table was rarefied to 28,000 reads per sample using the Vegan package v.2.6.4, with samples below this threshold excluded.
Institutions
- Nacionalni laboratorij za zdravje okolje in hrano
- Univerza v Mariboru Medicinska fakulteta