HerbsAuthenticate

The HerbsAuthenticate package process trimmed FASTQ files from both single or paired-end reads to perform sequence mapping against barcode databases. Additionally, it is also able to build customized databases based on the user’s needs by extracting specific barcodes from the trimmed reads.

Requirements and instalation

The script requires Python3 and the following programs to be installed:

• KMA
• blastn

To install the package, download the directory. Check the config.yaml file and make sure that all the paths to the executables are absolute and indeed refer to a working executable.

Indexing

Before aligning reads with KMA, the database must be indexed. This is done with the following command:

kma index -i path_to_database.fasta -o output_directory

The default k-mer size is 16. In case you want to change it, please add the option -k followed by the desired k-mer size.

Extract barcodes

The script Barcodes.py is used to obtain the barcodes from the trimmed reads. In order to execute it, please follow the steps indicated below:

1. Create an input file, using input.txt as an example. It is a space-separated file in which each line represents the data from one sample. There are three columns:

   • Column 1: absolute path to forward reads
   • Column 2: absolute path to reverse reads
   • Column 3: scientic sample name (Binomial nomenclature)

2. Ensure that config.yaml is located in the same directory as Barcodes.py and the scripts folder.

3. Update config.yaml parameters according to your data and type of analysis you want to perform. The options available are shown below:

   Path:

   • kma: Absolute path to KMA directory.
   • db_backbone: Absolute path to indexed backbone barcode database.
   • output: Absolute path to output directory.

   Validation:

   • merge_by_genus: boolean variable that indicates whether the validation is at genus or specie level. Default value: TRUE.

4. Excute the following command: python3 Barcodes.py input.txt

Mapping

To map trimmed reads against a specific barcode database, the script Mapping.py is used. In order to execute it, please follow the steps indicated below:

1. Create an input file, using input.txt as an example. It is a space-separated file in which each line represents the data from one sample. There are three columns:

   • Column 1: absolute path to forward reads
   • Column 2: absolute path to reverse reads
   • Column 3: sample name

2. Ensure that config.yaml is located in the same directory as Mapping.py and the scripts folder.

3. Update config.yaml parameters according to your data and type of analysis you want to perform. The options available are shown below:

   Path:

   • kma: Absolute path to KMA directory.
   • db: Absolute path to indexed database.
   • output: Absolute path to output directory.

   Reads:

   • PE/SE: Pair-ended (PE) or single-ended (SE) reads.

   Mapping:

   • merge_by_genus: boolean variable that indicates whether the identification is at genus or specie level. Default value: TRUE.
   • min_barcodes: Minimum number of identitifed barcodes from a genus or specie to be considered as a true positive. Default value: 2.

4. Excute the following command: python3 Mapping.py input.txt

For Computerome users

In case you are executing any of these scripts in Computerome:

1. Save either python3 Barcodes.py input.txt or python3 Mapping.py input.txt in a text file named run_HerbsAuthenticate_qsub.sh

2. Execute the following command:

qsub -W group_list=your_group -A your_group -l nodes=1:ppn=4,mem=50gb,walltime=1:00:00:00 -d path/to/your/output/directory run_HerbsAuthenticate_qsub.sh