DOI: 10.5061/dryad.jm63xsjm5
title: Comparison of fresh fecal samples from two populations (NAM and USA) (Part 2 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: # Comparison of fresh fecal samples from two populations (NAM and USA)
 (Part 2 of 2) Code for preprocessing FASTQ files and analysis to produce
 figures for Objective 2: fresh fecal comparison objective of "Fecal
 Microbiota is More Stable During Degradation and More Diverse for Ex Situ
 Cheetahs in Namibia Compared to the USA" ## Description of the data
 and files ### Coding files 1. Two.Pop.Comparisons_Preprocessing.Rmd - code
 for preprocessing unpaired FASTQ files for fresh samples from NAM and USA
 (dada2, decontam, build phylogenetic tree, create phyloseq files,
 calculate alpha diversity metrics) 2.Two.Pop.Comparisons_Analyses.Rmd -
 code for statistical tests and visualizations of relative abundance and
 alpha and beta diversity 2. CPTf23_Picrust2.ipynb - Jupyter Notebook file
 for running PICRUSt2 3. CPTf23_Picrust2_categorize_by_function.Rmd - code
 for merging and categorizing Picrust output into level 1, 2, and 3
 categories 4. CPTf23_ALDEx2_PICRUST.Rmd - code for statistical analyses
 and visualizations of picrust data at levels 1, 2, and 3 ## Files for
 phyloseq object (needed for Analyses code if not building from scratch) 1.
 CPTf23_featuretable_Nov23.csv - table of ASV counts by samples (first
 column has no header as is expected format for phyloseq) 2.
 CPTf23_taxonomy_Nov23.csv - mapping file for ASV numbers to taxonomic
 classifications (first column has no header as is expected format for
 phyloseq) ``` NAs in taxonomy file indicate corresponding taxonomy level
 was not identified for that ASV. ``` 1. Metadata_CPT23_final.csv -
 metadata for samples, see below table for explanation of columns |
 Row\.names | **Row names for importing data set** | |
 :----------------------- |
 :----------------------------------------------------------------------------------- | | **SampleID.x** | Sample ID names that match FASTQ sequences | | **FecalID** | Fecal Series sample belongs to | | **SampleDay** | Which day of the series it was collected | | **SampleDate** | Calendar day the fecal subsample was collected | | **quant\_reading** | DNA concentration of extraced DNA from fecal (before PCRs) as measured by flurometer | | **sample.type** | TRUE for fecal samples (FALSE for negative controls before filtering) | | **is.neg** | FALSE for fecal samples (TRUE for negative controls before filtering) | | **Sample\_or\_Control** | True Sample for fecal samples (Control for negative controls before filtering) | | **Population** | Which population the fecal sample came from (USA or NAM for Namibia) | | **PD** | Faith's Phylogenetic Diversity | | **SR** | Species Richness (ASV count) | | **SampleID.y** | Copy of rownames for metadata/sequencing matching | | **sample\_sums(CPTf23)** | sequence count per sample after decontam and filtering | 3. CPTf_2023_DNAsequences.csv - DNA sequences for all ASVs 4. CPTf23_cleanseqs_tree.nwk - phylogenetic tree of ASV sequences ## Sharing/Access information Raw FASTQ sequences used to generate these summary data files (and code) can be found: [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1160820](https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1160820) Other related datasets include: 1. Objective 1 (Part 1 of 2): DOI: 10.5061/dryad.sn02v6xdq 2. Separate manuscript where USA samples were originally published: (manuscript: [https://mah.bioscientifica.com/view/journals/mah/2/1/MAH-23-0022.xml](https://mah.bioscientifica.com/view/journals/mah/2/1/MAH-23-0022.xml) The code and data can also be found on GitHub: Objective 1: [https://github.com/Malytherin/CheetahFecalStability_Namibia](https://github.com/Malytherin/CheetahFecalStability_Namibia) Objective 2 (this data set): [https://github.com/Malytherin/Comparing.Fresh.Fecals_NAMvUSA](https://github.com/Malytherin/Comparing.Fresh.Fecals_NAMvUSA) USA manuscript data: [https://github.com/Malytherin/CheetahFecalStability_USA](https://github.com/Malytherin/CheetahFecalStability_USA)