Overview

FloRes (Flow Resistome) is the Bangor University & Compass Bioinformatics developed version of the AMR++ core repository with notable additions and modifications, and parameterised for running on different compute systems (including Super Computing Wales, Verily Workbench and Google Compute Cloud).

Notable changes to original pipeline:

• Additional integrated modules:

  • fastp
  • bowtie2 (host removal)
  • minimap2 (for longread data (Pacbio hifi/ONT))
  • bracken
  • R scripts for normalisation of amr tables

• Code changes and fixes

  • Reduce I/O for large data inputs
  • Work with new kraken2 taxonomy structure
  • Apptainer-slurm submission
  • Repair Nextflow channel bugs & version updates

  Note: Default alignment for megares is kept as BWA consistent with original AMR++ and can be changed by parameter. Bowtie2 alignment against megaresDB results in significant change on alignment rate.

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Preparing the install

1. Download the github repository with git clone and the url above

Containers

2. The pipeline is configured to run either with apptainer/singularity locally with automatic container download, or with docker if using google-batch remotely. You may choose to pre-build the singulariy images in advance if wanted (containers defined in conf/ folder) Note: If you are using Workbench/batch then you must pre-build the docker containers locally and upload to the container repository with the google CLI sdk.

Reference databases

3. Default downloads are performed on first run but you will likely want to download newer or more appropriate reference files

bowtie2 index files to remove contamination/host DNA

Either download directly, or build your indexes to be filtered against from user supplied fasta files. [Recommended] download human genome indexes directly from: https://bowtie-bio.sourceforge.net/bowtie2/index.shtml

Kraken2 database

The default parameter with AMR++ is to download the minikraken2 (2020) database which covers Bacteria, Archaea, Viruses which will occur the first time you run the pipeline. However you likely want a more updated version, and perhaps including fungi and plasmodium. They can be downloaded from here: https://benlangmead.github.io/aws-indexes/k2 and put in a location defined in the params.config file.

Slurm submission scripts if required

4. An example slurm script defines these parameters:
   • workdir: Location where processing will be performed (advice: use high speed location on your processing node i.e. /tmp)
   • installdir: Location of this github repo on your system
   • resultsdir: Where do you want the main outputs to be transfered to (not the full working folders & outputs)
   • run: Name of the sequencing run for annotation and folder where the fastq files are Default input read location is: $workdir/$run/fastq

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Running the pipeline

Default pipeline is standard_AMR_wKraken_and_bracken which will run from raw fastqs to the endpoint. Alternatives are to use mid-process data, kraken only etc. by modifying the --pipeline parameter

Core pipelines:
    - demo: Run a demonstration of AMR++
    - standard_AMR_wKraken: Run the standard AMR++ pipeline with Kraken
    - standard_AMR_wKraken_and_bracken: Run the standard AMR++ pipeline with Kraken AND Bracken
    - standard_hifi: Run the standard AMR++ pipeline for long-read (cleaned) data

Available subworkflows:
    - eval_qc: Run FastQC analysis
    - trim_qc: Run trimming and quality control
    - rm_host: Remove host reads
    - resistome: Perform resistome analysis
    - align: Perform alignment to MEGARes database
    - kraken: Perform Kraken analysis
    - **NEW** kraken_and_bracken: Perform Kraken and Bracken analysis
    - qiime2: Perform QIIME 2 analysis
    - bam_resistome: Perform resistome analysis on BAM files

You're finished!