Pipeline for automated translated BLAST searches in genomic sequences (tblastn-genomes)

Nextflow pipeline for automated translated BLAST searches in genomic sequences. For a broader context, see the following publication:

Bukowski M, Banasik M, Chlebicka K, Bednarczyk K, Bonar E, Sokołowska D, Żądło T, Dubin G, Władyka B. (2025) Analysis of co-occurrence of type II toxin–antitoxin systems and antibiotic resistance determinants in Staphylococcus aureus. mSystems 0:e00957-24. https://doi.org/10.1128/msystems.00957-24

This pipeline utilises CARD data (located in the input directory):

Alcock BP, Huynh W, Chalil R, Smith KW, Raphenya AR, Wlodarski MA, Edalatmand A, Petkau A, Syed SA, Tsang KK, Baker SJ. CARD 2023: expanded curation, support for machine learning, and resistome prediction at the Comprehensive Antibiotic Resistance Database. Nucleic acids research. 2023 Jan 6;51(D1):D690-9.

It is highly recommended to obtain the current data set from card.mcmaster.ca (Download/Download CARD Data). The pipeline utilises the protein homology model sequences.

Detailed comments on what and how each process do are provided in the main.nf file as well as in comments in the template files.

Values for technical parameters are defined in the nextflow.config file.

1. Environment

In envs/ directory YML files describe conda environments used by the pipeline. If you want to install the latest versions of the packages, remove version designations from all YML files (=X.X.*). The repository has been tested on Ubuntu 22.04 using conda 24.11.

Miniconda/Anaconda installation is a prerequisite. The tblastn-nextflow environment described in the tblastn-nextflow.yml file must be created prior running the pipepline. It utilises nexflow 23.10 package. Run the following command from the pipeline directory to create the environment:

conda env create -f envs/tblastn-nextflow.yml

3. Directory structure and pipeline files

tblastn-genomes/
├── envs/
│   ├── tblastn-nextflow.yml
│   ├── tblastn-blast.yml
│   └── tblastn-data.yml
├── input/
│   ├── antitoxins.faa
│   ├── toxins.faa
│   └── card.faa
├── templates/
│   └── filter.py
├── fna/
├── work/
├── output/
├── nextflow.config
└── main.nf

In the working directory you can find the main.nf file that describes the pipeline next to nextflow.config file describing the pipeline technical configuration. The envs/ directory contains YML files describing conda environments. In the input/ directory sets of protein query sequences are located. In the fna/ directory genomic sequences of analysed genomes are supposed to be located. The location of last two directories can be changed in the nextflow.config file. Template scripts are in template/. Directories work/ and output/ will be created automatically once the pipeline is run. The latter will contain final output from each stage of the analysis.

4. Pipeline architecture

The pipeline expects the following input files:

1. queries - a directory with protein FASTA files, each containing a set of sequences to be searched for corresponding coding sequences in analysed genomes. The file names must follow the pattern: .faa
2. genomes - a directory (here local directory fna) with genomic sequences, genomic sequence(s) for each genomes are in a gzipped FASTA file with the following NCBI GenBank name pattern: _genomic.fna.gz

The pipeline encompasses the following stages:

1. tblastnGenomes searches for sequences in analysed genomes that code for similar protein sequences to those from query sets.
2. addAssembly simply adds to BLAST search results a column with assembly accession version of the analysed genome, useful for downstream analyses (it links a match to a given genome).
3. filterRes filters the results in order to obtain a set of the best non-overlapping matches. At the end results for all genomes are merged into one file (output/tblastn_merged.tsv).

See comments in the template files for more details.

5. Running the pipeline

To start the pipeline activate the tblastn-nextflow environment and run Nextflow from the pipeline directory:

conda activate tblastn-nextflow
nextflow main.nf