DOI: 10.5281/zenodo.17649426
title: Supplementary Dataset and Workflows for 'Soil origin affects gut microbiota and immune response in a wild rodent'

description: Dataset and Workflows for "Soil origin affects gut microbiota and immune response in a wild rodent"


Description of the data and file structure


This dataset includes code to preprocess short read data deposited in ENA SRA (PRJEB87226) using QIIME2 and subsequent data analysis using R with related packages, processed 16S and ITS2 datasets required for the R pipeline, and reproducible nf-core workflow reports for RNA sequencing and differential expression analysis. Please see project GitHub repo for updates on scripts.


Files and variables


File: readme.md


Description: This file.


File: FECES_16S_QIIME2.sh


Description: Bank vole faecal 16S sequence data pre-processing pipeline

using QIIME2.


File: ITS2_QIIME2_vsearch.sh


Description: Bank vole faecal ITS2 sequence data pre-processing pipeline

using QIIME2.


File: dna-sequences_16S.fasta


Description: Denoised and decontaminated 16S V3-V4 DNA sequences

produced by QIIME2 pipeline.


File: dna-sequences_ITS2_c97.fasta


Description: Denoised and decontaminated 16S V3-V4 DNA sequences

clustered at 97% sequence identity produced by QIIME2 pipeline.


File: feature-table_ITS2_c97.tsv


Description: Decontaminated fungal ITS2 OTU abundance table produced by

QIIME2 pipeline.


File: feature-table_16S.tsv


Description: Decontaminated bacterial 16S ASV abundance table produced

by QIIME2 pipeline.


File: rooted-tree_16S.nwk


Description: Rooted phylogenetic tree for bacterial 16S data.


File: metadata_feces-soil_16S.txt


Description: Metadata file for 16S samples to be loaded in R.


Variables


SampleID: Sample ID


Sample_ID: Sample alias


Pup_ID: Subject ID


of the experimental animal


Sample_type: Sample type.


Sampling_timepoint: Sampling time pre- (T1) or post (T2) treatment.


is.neg: Identifier for negative control used in DECONTAM in QIIME2

pipeline.


pup_birth_date: Birth date of subject.


cage: Cage number of subject.


sex: Sex of subject.


Soil_location: City or national park from which experimental soil was

collected.


Soil_type: Main experimental factor - Control, Park or Urban.


Pup_head: Subject head width at birth


Pup_wt: Subject body weight at birth (g)


End_head: Subject head width at end of experiment (mm)


End_wt: Subject body weight at end of experiment (g)


Testis_wt: Left testis weight (g) (males only)


Sampling_date: Date of endpoint sampling


Mother_head: Head width of the subject's mother at subject's weaning

(mm).


Mother_wt: Body weight of the subject's mother at subject's weaning (g).


Mother_ID: ID of subject's mother. Used as random intercept in linear

mixed models to control for genetic component.


Mother_type: Information on subject's mother.


FEAST: Subsetting factor for sourcetracking using FEAST.


Env: Sample name in format required for FEAST source tracking.


SourceSink: Source or Sink population identifier for FEAST.


id: Sample identifier in format required for FEAST.


 

sample_accession: ENA sample accession ID



File: metadata_ITS2_c97.tsv


Description:Metadata file for ITS2 samples to be loaded in R.


Variables


As above.


File: taxonomy_ITS2_c97.guilds.txt


Description: Taxonomic classifications for ITS2 data, including FUNGuild

annotations.


File: taxonomy_16S.tsv


Description: Taxonomic classifications for 16S data.


File: firstdistances_bacteria_bray.tsv


Description: Table of Bray-Curtis first distances of faecal bacteria

pre- and post-treatment (=distance of the same subject between two time

points). Data produced by QIIME2 pipeline, to be plotted in R.


Variables


SampleID: Sample ID


SubjectID: Sample alias


Distance: Distance


Group: Experimental factor


File: firstdistances_bacteria_jaccard.tsv


Description: Table of Jaccard first distances of faecal bacteria pre-

and post-treatment (=distance of the same subject between two time

points). Data produced by QIIME2 pipeline, to be plotted in R.


Variables


As above


File: firstdistances_fungi_bray.tsv


Description: Table of Bray-Curtis first distances of faecal fungi pre-

and post-treatment (=distance of the same subject between two time

points). Data produced by QIIME2 pipeline, to be plotted in R.


Variables


As above


File: firstdistances_fungi_jaccard.tsv


Description: Jaccard first distances of faecal fungi pre- and

post-treatment (=distance of the same subject between two time points).

Data produced by QIIME2 pipeline, to be plotted in R.


Variables


As above


File: FEAST_F_contributions.txt


Description: Combined fungal source contributions from soil and food to

faecal communities. Data produced in

"faecal_metataxonomics_markdown.html" pipeline and externally edited in

excel. Used as input for FEAST figure. Data also available in excel file

Supplementary Data SI8.


Variables


Sample: Sample ID


Soil: Source contribution of soil mixture to which subject was exposed.


Food: Source contribution of food pellets.


Unknown: Unknown source component.


Group: Experimental factor


Location: Origin of soil mixture


File: faecal_metataxonomics_corestats.html


Description: Microbial diversity analyses in R markdown. All required input

files are published along this pipeline.


File: faecal_metataxonomics_FEAST.html


Description: Microbial source tracking analysis in R markdown. All required input

files are published along this pipeline.


File: faecal_metataxonomics_diffabund.html


Description: Microbial differential abundance analysis in R markdown. All required input

files are published along this pipeline.


File: nfcore_RNAseq.html


Description: Nextflow nf-core/RNAseq workflow report and settings for

preprocessing of bank vole colonic mRNA reads. Includes extensive

quality control and run settings.


File: nfcore_differentialabundance.html


Description: Nextflow nf-core/differentialabundance workflow report for

exploratory data analysis and differential expression analysis.


File: medoid_subsampling.R


Description: R script for unsupervised subsampling for RNA extraction

based on *a priori* knowledge of amplicon sequencing data.

 

 



File: soil2gut.network.R


Description: Script for microbial co-occurrence network analysis.



Code/software


Amplicon sequencing data preprocessing: QIIME2 distribution

qiime2-amplicon-2024.10. Preprocessing pipeline and settings are

described in Materials & Methods and files "FECES_16S_QIIME2.bash" and

"ITS2_QIIME2_vsearch.bash".


RNAseq data preprocessing and differential expression analysis: Data was

processed using nf-core/rnaseq v3.18.0 (doi:

[10.5281/zenodo.1400710](https://doi.org/10.5281/zenodo.1400710)) of the

nf-core collection of workflows ([Ewels *et al.*,

2020](https://doi.org/10.1038/s41587-020-0439-x)), utilising

reproducible software environments from the Bioconda ([Grüning *et al.*,

2018](https://doi.org/10.1038/s41592-018-0046-7)) and Biocontainers ([da

Veiga Leprevost *et al.*,

2017](https://doi.org/10.1093/bioinformatics/btx192)) projects. The

pipeline was executed with Nextflow v24.10.3 ([Di Tommaso *et al.*,

2017](https://doi.org/10.1038/nbt.3820)). Full details available in

"nfcore_RNAseq.html" and "nfcore_differentialabundance.html".


Data analysis: R 4.3.1. Analysis pipeline is available in R markdown

file "faecal_metataxonomics_markdown.html". All required input files are

included in this release. Required packages are displayed in the

markdown file.