Bacterial Genomics

General schematic of the steps in the workflow

Contents

• Quick Start
• Introduction
• Installation
• Usage
• Parameters
  • Required parameters
  • Optional parameters
  • Additional parameters
• Resource Managers
• Output
• Troubleshooting
• Contributions and Support
• Citations

Quick Start: Test

Run the built-in test set to confirm all parts are working as-expected. It will also download all dependencies to make subsequent runs much faster.

nextflow run \
  bacterial-genomics/wf-paired-end-illumina-assembly \
  -r main \
  -profile singularity,test

Quick Start: Run

Example command on FastQs in “new-fastq-dir” data using SPAdes with singularity:

nextflow run \
  bacterial-genomics/wf-paired-end-illumina-assembly \
  -r main \
  -profile singularity \
  --input new-fastq-dir \
  --outdir my-results \
  --assembler spades

Example command on FastQs in “new-fastq-dir” data using SKESA with singularity:

nextflow run \
  bacterial-genomics/wf-paired-end-illumina-assembly \
  -r main \
  -profile singularity \
  --input new-fastq-dir \
  --outdir my-results \
  --assembler skesa

Introduction

This workflow assembles bacterial isolate genomes from paired-end Illumina FastQ files. Post-assembly contig correction is performed, and a variety of quality assessment processes are recorded throughout the workflow.

This procedure can be used for all bacterial isolates (i.e., axenic, non-mixed cultures) sequenced with whole genome (WGS) or selective whole genome (SWGA) library preparation strategies. It is inappropriate for metagenomics analysis. The data files must be paired read sets (not single ended) and can come from any Illumina sequencing instrument which generates a FastQ file format (e.g., iSeq, HiSeq, MiSeq, NextSeq, NovaSeq).

The read set files can be obtained from an external source, local storage device, or sequencing instrument. Other sequencing manufacturers such as Ion Torrent, PacBio, Roche 454, and Nanopore generate data files that cannot be directly used with this procedure.

[!CAUTION] Due to the shorter DNA fragments of Illumina instruments, reads may be processed as singletons. It is recommended to check the DNA fragment length distribution using TapeStation or BioAnalyzer before sequencing.

Installation

• Nextflow >=21.10.3
• Docker or Singularity >=3.8.0
• Conda is currently unsupported

Usage

nextflow run \
  bacterial-genomics/wf-paired-end-illumina-assembly \
  -r main \
  -profile <docker|singularity> \
  --input <input directory|samplesheet> \
  --outdir <directory for results> \
  --assembler <spades|skesa>

Please see the usage documentation for further information on using this workflow.

Parameters

Note the “--” long name arguments (e.g., --help, --input, --outdir) are generally specific to this workflow’s options, whereas “-” long name options (e.g., -help, -latest, -profile) are general nextflow options.

These are the most pertinent options for this workflow:

Required parameters

  ============================================
        Input/Output
  ============================================
  --input                 Path to input data directory containing FastQ assemblies or samplesheet.
                          Recognized extensions are: .fastq and .fq with optional gzip compression (.gz)

  --outdir                The output directory where the results will be saved.

  ============================================
        Container platforms
  ============================================
  -profile singularity    Use Singularity images or auto-convert Docker images to run the workflow.

  -profile docker         Use Docker images to run the workflow.

  ============================================
        Optional assemblers
  ============================================
  --assembler             Specify which assembler to execute (spades, skesa). [Default: spades]

  ============================================
        Reference files
  ============================================
  --phix_reference        Path to PhiX reference file in FastA format.
                          Recognized extensions are: (fasta|fas|fa|fna). [Default: PhiX_NC_001422.1.fasta]

  --adapters_reference    Path to adapter reference file in FastA format. Recognized extensions are: (fasta|fas|fa|fna).
                          [Default: dapters_Nextera_NEB_TruSeq_NuGEN_ThruPLEX.fas]

PhiX reference NC_001422.1 can be obtained from NCBI.

Optional parameters

  ============================================
        Optional databases
  ============================================
  --kraken1_db          Path to a local directory, archive file, or a URL to a compressed tar archive
                        that contain files `database.{idx,kdb}` and `taxonomy/{names,nodes}.dmp`.
                        [Default: MiniKraken 8GB]

  --kraken2_db          Path to a local directory, archive file, or a URL to a compressed tar archive
                        that contain `{hash,opts,taxo}.k2d` files.
                        [Default: Kraken2 Standard 8GB]

  --blast_db            Path to a local directory, archive file, or a URL to a compressed tar archive
                        that contains BLAST 16S ribosomal RNA files.
                        [Default: NCBI's 16S ribosomal RNA database]

  --gtdb_db             Path to a local directory, archive file, or a URL to a compressed tar archive
                        that contains the GTDB database.
                        [Default: NaN]

  --busco_db            Path to a local directory, archive file, or a URL to a compressed tar archive
                        that contains BUSCO lineages. Can either be a lineage dataset or entire BUSCO database.
                        [Default: NaN]

[!NOTE] If user does not specify inputs for parameters with a default set to NaN, these options will not be performed during workflow analysis.

Additional parameters

View help menu of all workflow options:

nextflow run \
  bacterial-genomics/wf-paired-end-illumina-assembly \
  -r main \
  --help \
  --show_hidden_params

Resource Managers

The most well-tested and supported is a Univa Grid Engine (UGE) job scheduler with Singularity for dependency handling.

1. UGE/SGE
   • Additional tips for UGE processing are here.
2. No Scheduler
   • It has also been confirmed to work on desktop and laptop environments without a job scheduler using Docker with more tips here.

Output

Please see the output documentation for a table of all outputs created by this workflow.

Troubleshooting

Q: It failed; how do I find out what went wrong?

A: View file contents in the <outdir>/pipeline_info directory.

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.