Wild again: Recovery of a beneficial Cannabis seed endophyte from low domestication genotypes

Authors: Carolina Lobato, João Machado de Freitas, Daniel Habich, Isabella Kögl, Gabriele Berg & Tomislav Cernava

Institute of Environmental Biotechnology (UBT) — TU Graz

Project structure

project/
├── data/
│   └── metadata/
│       ├── CB1/
│       │   ├── bcpr-fw.fasta
│       │   ├── bcpr-rv.fasta
│       │   ├── cat.sh
│       │   └── manifest.csv
│       ├── CB2/
│       │   ├── bcpr-fw.fasta
│       │   ├── bcpr-rv.fasta
│       │   ├── cat.sh
│       │   └── manifest.csv
│       ├── CB3/
│       │   ├── bcpr-fw.fasta
│       │   ├── bcpr-rv.fasta
│       │   ├── cat.sh
│       │   └── manifest.csv
│       ├── metadata.csv
│       ├── pouches-ind.tsv
│       ├── pouches-exp.tsv
│       ├── field23.tsv
│       └── PLaBase.tsv
├── scripts/
│   ├── bash/
│   │   ├── QIIME2.sh
│   │   └── WGS.sh
│   ├── R/
│   │   ├── Setup.Rmd
│   │   ├── Figure1.Rmd
│   │   ├── Figure2.Rmd
│   │   ├── Figure3.Rmd
│   │   ├── Figure4.Rmd
│   │   └── Figure5.Rmd
│   └── utils/
│       ├── csv2fasta.sh
│       ├── csv2tsv.sh
│       ├── QIIME22R.sh
│       ├── install.R
│       ├── plot_composition_v2.R
│       ├── UMAP/
│       │   ├── data.R
│       │   ├── project.R
│       │   ├── cluster-analysis.R
│       │   └── run-all.R
│       └── biomarkers/
│           ├── data.R
│           ├── feature-importance.R
│           ├── sv-importance-patch.R
│           ├── train-eval.R 
│           └── run-all.R
├── outputs/
│   ├── QIIME2/
│   └── R/
├── README.md
└── LICENSE

Details

• data/

  • metadata/ This subdirectory contains:
    • Barcode fasta files for each pool (bcpr-fw.fasta and bcpr-rv.fasta).
    • Concatenating files for each pool (cat.sh).
    • Manifest files for each pool (manifest.csv).
    • Sample information for the metabarcoding analysis (metadata.csv).
    • Metadata and measurements at individual-level in lab trials (pouches-ind.tsv)
    • Metadata and measurements related to each replicate experiment in lab trials (pouches-exp.tsv)
    • Metadata and measurements of the field trials (field23.tsv)
    • Output gene class table from PLaBase (PLaBase.tsv)

• scripts/

  • bash/ This subdirectory contains the scripts used for:

    • Amplicon sequencing processing with QIIME2 v2023.5, including:
      1. Demultiplexing with CUTADAPT v4.2
      2. Importing into QIIME2
      3. Bioinformatic processing steps using the DADA2 pipeline and VSEARCH with the SILVA v138 reference database to generate the feature table, taxonomy file, representative sequences, and the phylogenetic tree
      4. Exporting from QIIME2
    • Genome assembly of Bacillus frigoritolerans using FLYE v2.9.3 and annotation with DRAM

  • R/ This subdirectory contains the scripts used in R to create the phyloseq objects, preprocess the data, and prepare the figures for the manuscript.

  • utils/ This subdirectory contains utility scripts that are used by other scripts in the project, such as:

    • csv2fasta.sh for converting .csv to .fasta format.
    • csv2tsv.sh for converting .csv to .tsv format.
    • QIIME22R.sh for converting .biom to .tsv format and back,
    • install.R for installing the necessary packages in R.
    • plot_composition_v2 modified microbiome::plot_composition function for running when the microbiome version is above 1.6.
    • UMAP/ contains the scripts used for beta diversity representation with UMAP, as shown in Figure 2.
    • biomarkers/ contains the scripts used for biomarker assessment shown in Figure 3.

• outputs/ contains QIIME2 and R saved outputs.

Further content

The 16S rRNA gene amplicon raw FASTQ files were deposited in ENA under the accession number PRJEB64469.

The assembled genome of Bacillus frigotolerans, with the associated annotations, was deposited in NCBI under accession number PRJNA1113337.

References

Lobato, C. et al. Wild again: recovery of a beneficial Cannabis seed endophyte from low domestication genotypes. Microbiome 12, 239 (2024). https://doi.org/10.1186/s40168-024-01951-5