5 Juno-amr

The Juno Antimicrobial Resistance(Juno AMR) pipeline is a pipeline that is used to automate multiple tools that help identify acquired genes and find chromosomal mutations mediating antimicrobial resistance in DNA sequences of bacteria. The tool takes paired-end .fastq files as input. The input files can contain full or partial sequences. The output can be used in antimicrobial resistance analysis and is combined in four summary files for a quick overview of the most important results.

The tools that are being used in this pipeline are listed below:

• ResFinder is a tool that identifies acquired antimicrobial resistance genes based on databases. The tool is created by The Center For Genomic Epidemiology.

• PointFinder is a tool that searches for chromosomal point mutations that mediate resistance to select antimicrobial agents for some bacterial species. The tool is created by The Center For Genomic Epidemiology.

5.1 Handbook

5.1.1 Requirements and preparation

See the General Instructions for all pipelines first.

To run this pipeline the type of species and a folder with paired .fastq files is required.

Place all your samples in one folder, no subfolders, all file types should be the same format.

Besides giving input it is also required to select a species. The --species parameter is required for PointFinder in order to find the chromosomal mutations. If you don’t know the species you can fill this parameter with ‘other’. This way the pipeline will run ResFinder without running PointFinder.

The output of the pipeline will be collected in the folder ‘output’ within the working directory (the directory where the pipeline is active). If you want the output to be in a different location it is possible to do this with the --output parameter.

Note: all samples in the directory should be the same species and the same file format.

5.1.2 Download the pipeline

YOU NEED TO DOWNLOAD THE PIPELINE ONCE OR EVERY TIME YOU WANT TO UPDATE IT

Make sure to have followed the instructions to set up conda before installing any of our pipelines!

Please follow the instructions to download pipelines from the Juno team of the IDS-bioinformatics group.

The Juno-amr pipeline can be found here.

5.1.3 Start the analysis. Basics

1. Open a terminal. (Applications>terminal).

2. Enter the folder of the pipeline using:

cd /mnt/scratch_dir/<my_folder>/Juno-amr

3. Run the pipeline
   

python3 juno-amr.py -s [species] -i [/mnt/scratch_dir/<my_folder>/fastq_file folder]

Please read the section What to expect while running a Juno pipeline.

See the section General Troubleshooting for any problems you may encounter.

Note: Do not keep all your data (including results) on the scratch_dir partition. You can add the --output parameter to the command in order to place your output somewhere else.

5.1.4 Output

In the output you can find 3 folders:

• log: Log with output and error file from the cluster for each snakemake rule/step that is performed.

• results_per_sample: Output produced by ResFinder and PointFinder for each sample. CGE’s explanation on the output can be found here.

• summary: Directory with 4 summary files created from each sample within the results_per_sample folder.

  • summary_amr_genes.csv: Shows the samplename resistance gene, identity, alignment length/gene length and coverage per gene hit.

  • summary_amr_phenotype.csv: Shows the type of match for each sample on the resistance for each antimicrobial.

  • summary_amr_pointfinder_predicition.csv: - Shows the samplename and a 0(no hit) or 1(hit) prediciton for each mutation.

  • summary_amr_pointfinder_results Shows the samplename, mutation, nucleotide change, amino acid change, resistance and PMID for each mutation hit

Note: If you want your output to be stored in a folder with a different name or location, you can use the option -o or --output

python3 juno-amr.py -i /mnt/scratch_dir/<my_folder>/<my_data>/ -species salmonella -o /mnt/scratch_dir/<my_folder>/<my_results>/

5.1.5 Troubleshooting for this pipeline

Please read the General Troubleshooting section first!

5.1.5.1 Other problems or failing rules

The Juno-amr pipeline is still in development which means that sometimes the process can fail.

Before contacting for help, try these two steps:

1. Re-run the pipeline again and see if the process continues. If it does, please keep re-running the pipeline until your analysis is finished or there is no longer progress. In this case, send an email after the pipeline is finished so I can troubleshoot the problem.

2. Download the pipeline again and start from the beginning of this handbook. Sometimes there is an issue that has been resolved in newer versions of the pipeline.

If the pipeline still fails after these two steps, please inform me about the problem. Send an e-mail with the following content:

• The log and error files that can be found in the output folder
• The path to your input directory
• The path to where the pipeline is installed

Note: I cannot help you without this information, if information is missing there will be a delay in troubleshooting the problem.