:rocket: ASTRO: AMR Metagenomics Detection, Simulation, Taxonomic Classification, and Read Optimization

Introduction

ASTRO: AMR Metagenomics Detection, Simulation, Taxonomic Classification, and Read Optimization is a bioinformatics pipeline that performs taxonomic profiling, screens metagenomes and isolate genomes for determinants of antimicrobial resistance, simulates reads, and generates a bacterial metagenomic in silico reference dataset.

The three primary objectives of the ASTRO workflow entail:

• Simulate sequencing reads with identified species and phyla of interest
• Perform taxonomic profiling and antimicrobial resistance gene (ARG) detection on empirical metagenomes and simulated reads
• Verify that the quality of simulated datasets mimics empirical datasets

This workflow is being built with Nextflow DSL2 and utilizes docker and singularity containers to modularize the workflow for optimal maintenance and reproducibility.

Pipeline Summary

1. Input paired-end metagenomic reads (.fastq) and isolate genomes (.fna)
2. Perform preprocessing on metagenomic reads (FastQC, FastP, BBDuk, Hostile)
3. Assemble the preprocessed reads into contigs and assess the quality of the assembled contigs (MEGAHIT, QUAST)
4. Screen metagenomes for ARGs (AMRFinderPlus, ABRICATE, RGI)
5. Perform taxonomic profiling on metagenomic reads to identify microbial community composition (METAPHLAN)
6. Simulate next generation sequencing reads and spike into cleaned, empirical metagenomic dataset (NEAT, RAGTAG)
7. Perform quality control (QC) on simulated dataset (FastQC)
8. Optionally perform taxonomic profiling and ARG detection on in silico dataset
9. Generate versions and MultiQC reports

Usage

Note If you are new to Nextflow and nf-core, please refer to this page on how to set up Nextflow. Make sure to test your setup with -profile test before running the workflow on actual data.

To run the astro pipeline minimal test, you will need to add your user-specific credentials for the --ncbiemail, --ncbiapi_key, and --metaphlan parameters to the profile script located at conf/test.config.

You can access the test data here.

Once complete, you can run the minimal test with the following command: nextflow run main.nf -profile test,singularity --outdir <OUTDIR>

Set Up:

First, input your metagenomic data into a samplesheet so that it resembles the following:

samplesheet.csv:

csv sample,fastq_1,fastq_2 Sample1,assets/data/ERR4678562_1.fastq.gz,assets/data/ERR4678562_2.fastq.gz Sample2,assets/data/ERR4678563_1.fastq.gz,assets/data/ERR4678563_2.fastq.gz

Each row represents a pair of fastq files (paired-end metagenomics reads).

You will also need to prepare a samplesheet for isolate genomes to be used in simulation:

isolate_samplesheet.csv:

csv sample_id,added_copy_number,file_path,species_name GCA_018454105.3,1,assets/data/GCA_018454105.3_PDT001044797.3_genomic.fna,Acinetobacter baumannii GCA_016490125.3,1,assets/data/GCA_016490125.3_PDT000725303.3_genomic.fna,Acinetobacter baumannii

Each row corresponds to the following information:

• sample_id: Sample ID or name

• added_copy_number: Option to include a given number of copies of simulated genomes. If copy number variation is not desired, input '1'

• file_path: Path to isolate genome file (.fna)

• species_name: Name of isolate species

For instructions on creating an NCBI account and obtaining an API key, please visit the National Library of Medicine Support Center.

Taxonomic Classification Set Up

For ASTRO >= 1.0.0, you are required to install the MetaPhlAn 4 database. You can find guidance to do so on the MetaPhlAn GitHub page. It is highly recommended to use the --index parameter to download a specific version of the database for use in the ASTRO pipeline. This version of ASTRO was tested using database version mpavJun23CHOCOPhlAnSGB_202403.

ASTRO Pipeline Parameters:

• --input: Input metagenomic samplesheet

• --isolates: Input isolate samplesheet

• --ncbi_email: User's NCBI email

• --ncbi_api_key: User's NCBI API key

• --postsim: Optionally run assembly, AMR, and taxonomic classification on simulated reads

• --mode: Select 'local' if providing local paths to isolate genomes; otherwise, select 'download'

• --taxadb: Path to MetaPhlAn database

Running ASTRO:

Now, you can run the pipeline using:

```bash nextflow run main.nf \ --input samplesheet.csv \ --isolates isolatesamplesheet.csv \ --ncbiemail \ --ncbiapikey \ --postsim \ -profile singularity \ --outdir \ --mode or \ --taxadb $PATHTODB \

```

Note that --postsim is an optional parameter. If used, simulated data will be processed for assembly, ARG detection, and taxonomic classification.

Warning:** Please provide pipeline parameters via the CLI or Nextflow -params-file option. Custom config files, including those provided by the -c Nextflow option, can be used to provide configuration; see docs.

Credits

ASTRO was originally written by the Next Generation Sequencing (NGS) Quality Initiative (QI) In Silico Team.

We thank the following partners for their extensive assistance in the development of this pipeline:

• CDC Clinical and Environmental Microbiology Branch (CEMB)
• CDC Office of Advanced Molecular Detection (OAMD)
• CDC Office of Laboratory Systems and Response (OLSR)
• CDC Division of Laboratory Systems (DLS)

Contributions and Support

If you would like to contribute to this pipeline, please see the contributing guidelines.

Citations

An extensive list of references for the tools used by the pipeline can be found in the CITATIONS.md file.

This pipeline uses code and infrastructure developed and maintained by the nf-core community, reused here under the MIT license.

The nf-core framework for community-curated bioinformatics pipelines.

Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen.

Nat Biotechnol. 2020 Feb 13. doi: 10.1038/s41587-020-0439-x.

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