Introduction

MontGen is a bioinformatics best-practice analysis pipeline for bacterial and small eukaryote assembly and annotation. This pipeline is derived from the nf-core/bacass pipeline, which assembles short reads, long reads, or a mixture of both. Key differences between MontGen and nf-core/bacass are highlighted below.

The pipeline is built using Nextflow, a workflow tool that runs tasks across multiple compute infrastructures in a portable manner. It uses Docker/Singularity containers, making installation straightforward and results highly reproducible. The Nextflow DSL2 implementation uses one container per process, simplifying software dependency management. Where possible, processes have been submitted to and installed from nf-core/modules, making them available to the Nextflow community.

Pipeline Output

MontGen extends the nf-core pipeline by adding modules that use reference genomes and annotations to generate metrics and reference-based assemblies. For a full list of output files, see the documentation.

The subworkflow includes the following processes after aligning against the reference genome with minimap2:

1. Calculate reference genome coverage.
2. Identify missed regions in the reference genome.
3. Generate a coverage track for visualization.
4. Create an ASM Dot-Plot for assembly and reference genome comparison using pafCoordsDotPlotly.
5. Perform gene annotation with Liftoff (Shumate et al.).
6. Detect structural variants (e.g., deletions, insertions, inversions) with bcftools and format results into a user-friendly table using cuteSV.

Note: These steps will happen if the --reference_gff and --reference_fasta are provided.

Pipeline Summary

MontGen has been extensively tested with nanopore data alone or in combination with short reads. Commonly used options include:

• Dragonflye for nanopore-only or hybrid data.
• Miniasm for nanopore-only data.

Usage

Note: If you are new to Nextflow and nf-core, refer to this guide for setup instructions. Test your setup with -profile test before running the workflow on actual data.

Prepare a samplesheet with your input data as follows:

samplesheet.tsv:

ID              R1                      R2                      LongFastQ           Fast5   GenomeSize
shortreads      ./data/S1_R1.fastq.gz   ./data/S1_R2.fastq.gz   NA                  NA      NA
longreads       NA                      NA                      ./data/**fastq.gz   NA      2.8m
shortNlong      ./data/S1_R1.fastq.gz   ./data/S1_R2.fastq.gz   ./data/**fastq.gz   NA      2.8m

Each row represents a single-end or paired-end fastq file.

Note: MontGen supports nanopore multi-file input but has not been tested with FAST5 files.

Example of long-read assembly with Miniasm:

nextflow run nf-core/bacass -profile docker --input samplesheet.tsv
--assembly_type 'long' --assembler 'miniasm' --outdir <OUTDIR>
--reference_fasta <REFERENCE.FASTA> --reference_gff <REFERENCE.GFF>

Note: New modules are supported only with Docker containers. Specify the Docker profile to use these modules. Containers are hosted on Seqera Containers.

Warning: Provide pipeline parameters via the CLI or the Nextflow -params-file option. Custom config files (e.g., those provided with the -c option) can configure everything except parameters. See the documentation for details.

Credits

The nf-core/bacass pipeline was initiated by Andreas Wilm, originally written by Alex Peltzer (DSL1), rewritten by Daniel Straub (DSL2), and maintained by Daniel Valle-Millares.

MontGen was developed by Lorena Pantano and Alex Bartlett.

Additional contributors:

• Lorena Pantano
• Alex Bartlett
• Alkmini Diamantidi (alkmini.diamantidi@braskem.com)
• Hugo Rody Vianna Silva (hugo.vianna@braskem.com)
• Wing-On (Jacky) Ng (wingon.ng@braskem.com)

Citations

If you use nf-core/bacass for your analysis, cite it using the following DOI: 10.5281/zenodo.2669428.

For a comprehensive list of references for tools used in the pipeline, see the CITATIONS.md file.

You can cite the nf-core framework as follows:

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.

Future Development

To make MontGen a fully compatible nf-core pipeline, the following tasks need to be completed:

1. Convert all new modules listed in the Pipeline Output section into nf-core modules. This includes:

   • ASM Dot-Plot
   • Liftoff
   • bcftools
   • cutesv

2. Ensure all modules adhere to nf-core guidelines and standards.

3. Submit the modules to the nf-core/modules repository for community use.

4. Update the documentation to reflect the integration of these modules into nf-core.

Finally, adapting test data to the new parameters.

By completing these tasks, MontGen will align with nf-core's modular framework, enhancing its usability and maintainability within the Nextflow community.