DOI: 10.5061/dryad.sn02v6xdq
title: Fecal microbiota degradation over time in ex situ Namibian Cheetahs (Part 1 of 2)

description: The relationships between gut microbiota and animal health are an
 important consideration that are increasingly influential in the
 management of wild and ex situ endangered species, such as the cheetah
 (Acinonyx jubatus). To better understand these relationships, fresh fecal
 samples are currently required as a non-invasive alternative for the gut
 microbiome. Unfortunately, fresh samples are challenging to collect,
 especially in the wild. This study had two main aims: 1) to determine the
 optimal collection time point for cheetah feces after deposit in their
 native environment of Namibia as a guide for wild cheetah fecal microbiome
 studies; and 2) to characterize the cheetah fecal microbiota between two
 ex situ cheetah populations from two locations (Front Royal, VA, USA and
 Otjiwarongo, Namibia), which also consume different diets (commercial diet
 + carcass supplements and carcass only, respectively). Fresh fecal samples
 were collected from 16 cheetahs (Virginia, n = 8; Namibia n = 8) and
 analyzed for bacterial community diversity and composition using 16S rRNA
 gene amplicon sequencing. First, we found no difference in bacterial
 richness, diversity, or community composition from fresh fecal material
 compared to subsequent samples of decomposing fecals over a four-day
 sampling period in Namibia. Second, fresh cheetah samples in semi-wild
 conditions in Namibia had higher number of bacterial taxa, more
 phylogenetically diverse bacteria and contained compositionally distinct
 microbiomes (Bray-Curtis dissimilarity, Jaccard, and unweighted UniFrac
 measures) from cheetahs in zoo-managed conditions in the USA. We also
 found few differences in predictive functions of the fecal microbiota
 between the populations, where only one disease-related pathway was higher
 in the USA samples. Overall, our findings suggest that in dry season
 conditions (no recorded rainfall) in Namibia, fecals may be usable for up
 to three days after defecation for microbial ecology studies. We provide
 observational data on how to identify fecals within four days of being
 voided (see supplemental photos). There are major demographic and dietary
 differences between ex situ Namibian and USA populations, and we suggest
 further investigation into the influence of diet and population on the gut
 microbiota and health of cheetahs.
description: # Fecal microbiota degradation over time in ex situ Namibian Cheetahs
 (Part 1 of 2) ## Description of the data and file structure Code and files
 for preprocessing and analyses of 16S fecal microbiota data from cheetahs.
 Fecal samples were collected fresh and analyzed for an additional 4 days
 to observe microbial changes in fecals exposed to the environment. This
 data is for Objective 1 in the article: "Fecal Microbiota is More
 Stable During Degradation and More Diverse for *Ex Situ* Cheetahs in
 Namibia Compared to the USA" Data from the same article for Objective
 2 can be found: DOI: 10.5061/dryad.jm63xsjm5 ### R Markdown Files 1.
 NAM_Degradation_Preprocessing.Rmd - code for preprocessing unpaired FASTQ
 files (dada2, decontam, rarefactin curve, alpha calculations, create
 phyloseq object files) 2. NAM_Degradation_Analyses.Rmd - code for
 statistical tests and visualizations for relative abundance, alpha and
 beta diversity 3. NAM_Degradation_MaAslin2_Code.Rmd - code for running
 differential abundance testing across samples days using MaAslin2 ## Files
 for building phyloseq object 1. CCF8_featuretable_July2024.csv (feature
 table - ASV counts for each sample, first column are row names so there is
 no data in column 1, row 1 as is expected format for phyloseq processing)
 2. CCF8_Meta_July2024.csv (metadata for samples) | SampleID | **Full name
 of sample to match FASTQ file.** | | :--------------------------- |
 :---------------------------------------------------------------------------------------------------------- | | **Fecal.ID** | Series ID | | **SampleDay** | The day in the series it was collected | | **SampleDate** | Actual calendar day of fecal sampling. | | **quant.reading** | DNA concentration of extracted DNA (before any PCR) as measured by a fluorometer Used in decontam. | | **sample.type** | TRUE or FALSE. FALSE is for negative controls. Used in decontam. | | **is.neg** | TRUE or FALSE. TRUE is negative controls. Used in decontam and to separate out negatives from true samples. | | *Sample*\_or\_Control\*\* | TRUE sample = all fecal samples are true samples. | | **sample** | Full combination of Fecal ID and Sample Day using NAM moniker instead of CCF | | **PD** | Faith's Phylogenetic Diversity | | **SR** | Species Richness (ASV count) | | **sample\_sums.ccfclean24.** | number of sequences in each sample AFTER decontam and filtering | 1. CCF8_taxonomy_July2024.csv (taxonomy file for mapping ASV numbers to taxonomy classifications, first column has no header as expected format for phyloseq processing) 1. NAs: when NAs are present it indicates the corresponding column name/level could not be identified 2. CCFPT_2024_DNAsequences.fasta (Sequences of ASVs) 3. CCFPT_2024_tree.nwk (phylogenetic tree of DNA sequences from ASVs) ### Sharing/Access information: Raw sequence reads can be found in the SRA directory: [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1160820](https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1160820) Code and files can also be found on GitHub: ``` Objective 1: https://github.com/Malytherin/CheetahFecalStability_Namibia Objective 2: https://github.com/Malytherin/Comparing.Fresh.Fecals_NAMvUSA ```