sequana-denovo 0.12.0

Multi-sample denovo assembly of FastQ sequences (short read)

This is the denovo pipeline from the Sequana project.

Overview:

De-novo assembly pipeline for short-read Illumina data (bacterial genomes)

Input:

A set of paired or single-end FastQ files

Output:

Assembled FASTA contigs, annotation (GFF/GenBank), variant calls (VCF), HTML reports

Status:

Production

Documentation:

This README and https://sequana.readthedocs.io

Citation:

Cokelaer et al, (2017), ‘Sequana’: a Set of Snakemake NGS pipelines, Journal of Open Source Software, 2(16), 352, https://doi.org/10.21105/joss.00352

Installation

If you already have all requirements, install the package with pip:

pip install sequana_denovo --upgrade

You will need third-party tools (spades, prokka, quast, etc.). Install all dependencies at once:

mamba env create -f environment.yml

Usage

Scan FastQ files in a directory and set up the pipeline (replace DATAPATH with your input directory):

sequana_denovo --input-directory DATAPATH

To skip Prokka annotation:

sequana_denovo --input-directory DATAPATH --skip-prokka

To tune SPAdes memory (default 64 Gb) and digital normalisation:

sequana_denovo --input-directory DATAPATH --spades-memory 32 --digital-normalisation-max-memory-usage 1e9

This creates a denovo/ directory with the pipeline and configuration file. Execute the pipeline locally:

cd denovo
sh denovo.sh

If you are familiar with Snakemake, you can also run the pipeline directly:

snakemake -s denovo.rules --cores 4 --stats stats.txt

See .sequana/profile/config.yaml to tune Snakemake behaviour (cores, cluster settings, etc.).

Usage with apptainer

With apptainer, initiate the working directory as follows:

sequana_denovo --input-directory DATAPATH --use-apptainer

Images are downloaded in the working directory. To store them in a shared location:

sequana_denovo --input-directory DATAPATH --use-apptainer --apptainer-prefix ~/.sequana/apptainers

Then run as usual:

cd denovo
sh denovo.sh

Requirements

This pipeline requires the following executables (install via bioconda/conda):

• spades or unicycler — de-novo assembler (--assembler option)

• khmer — digital normalisation (normalize-by-median.py, filter-abund.py, etc.)

• quast — assembly quality assessment

• prokka — genome annotation (optional, --skip-prokka)

• busco — assembly completeness assessment (optional)

• checkm-genome — genome completeness and contamination (optional)

• bwa + sambamba — read mapping back to assembly

• freebayes — variant calling

• samtools — BAM/SAM processing

• seqkit — contig filtering by length

• blast — taxonomic identification of contigs (optional)

• multiqc — aggregated HTML report

• graphviz — pipeline DAG image

Details

This Snakemake pipeline assembles bacterial (or other small) genomes from short Illumina reads.

Digital normalisation (khmer): optionally reduces sequencing depth to a target coverage level, discarding redundant reads. This lowers memory usage and speeds up assembly without significantly impacting quality.

Assembly: SPAdes (default) or Unicycler. SPAdes uses multiple k-mer sizes and is recommended for most bacterial genomes. Unicycler is designed for hybrid or circular assemblies.

Quality assessment (QUAST): reports assembly statistics (N50, # contigs, total length, GC%, coverage depth) with an interactive Icarus contig browser.

Annotation (Prokka): rapid prokaryotic genome annotation producing GFF, GenBank, and other standard formats.

Coverage analysis (sequana_coverage): reads are mapped back to the assembly with BWA, duplicates flagged with Sambamba, and per-contig coverage profiles computed and visualised.

Variant calling (Freebayes): detects SNPs and small indels between the assembled consensus and the mapped reads.

Completeness (BUSCO / CheckM): optionally assess assembly completeness against conserved single-copy orthologs (BUSCO) or lineage-specific marker genes (CheckM).

Taxonomic identification (BLAST): optionally BLASTs the top contigs against the nt database to identify their taxonomy.

A summary HTML report (summary.html) with per-sample assembly statistics and embedded coverage plots is generated at the end of the run, alongside a MultiQC report.

Rules and configuration details

See the latest documented configuration file for all available parameters.

Changelog

Version	Description
0.12.0	Drop Python 3.8/3.9; require Python >=3.10 Drop click-completion dependency Replace pkg_resources with importlib.metadata in __init__.py Add exclude_pattern to config and schema Fix digital_normalisation: use khmer:2.1.1 container from damona (zenodo record 13924243) instead of generic sequana_tools image Fix digital_normalisation rule: convert run: to shell: for apptainer compatibility (container: + run: disallowed) Fix spades, prokka, unicycler rules: add named input.fastq / input.assembly for wrapper compatibility Fix bwa_index: add options param; bwa: drop tmp_directory, add options/resources params; new bwa/align v2 shell that separates bwa mem and sambamba sort into distinct steps Fix sambamba_markdup/sambamba_filter: add missing params/resources Fix samtools_depth: add named input.bam and options param Fix prokka output: .gff instead of .gbk (matches wrapper) Improved HTML report: per-sample summary.html with quast stats table and embedded coverage plots; main table links to per-sample reports; workflow=False on individual reports Update environment.yml: add khmer, python>=3.10 Update tools.txt: add khmer scripts, graphviz
0.11.1	Fix missing resources for quast/prokka/bwa_index
0.11.0	add checkm
0.10.0	use click / include multiqc apptainer
0.9.0	Major refactoring to include apptainers, use wrappers
0.8.5	add multiqc and use newest version of sequana
0.8.4	update pipeline to use new pipetools features
0.8.3	fix requirements (spades -> spades.py)
0.8.2	fix readtag, update config to account for new coverage setup
0.8.1
0.8.0	First release.

Contribute & Code of Conduct

To contribute to this project, please take a look at the Contributing Guidelines first. Please note that this project is released with a Code of Conduct. By contributing to this project, you agree to abide by its terms.