smk-bac-assembly

Snakemake Bacterial Assembly Workflow

A Snakemake workflow for Bacterial Genome assembly and QC using PacBio HiFi reads from University of Bern NGS Platform.

Overview

This workflow automates the complete pipeline for bacterial genome assembly:

1. QC on raw reads - NanoPlot generates comprehensive reports on sequencing data
2. Assembly - Flye assembler produces high-quality long-read assemblies
3. Assembly QC - Seqkit generates assembly statistics and summary reports
4. Completeness assessment - BUSCO evaluates assembly completeness against conserved orthologs
5. Taxonomic validation - PGAP performs taxonomic classification and validation
6. Genome annotation - Bakta provides comprehensive prokaryotic genome annotation

Features

• Modular design with separate rule files for each step
• Conda environment management for reproducibility
• Docker containerization for BUSCO and PGAP
• Comprehensive logging and output organization
• Automated sample discovery from CSV file
• Support for batch processing multiple samples
• Completeness assessment with BUSCO
• Taxonomic validation with PGAP
• Genome annotation with Bakta
• Summary tables for QC and taxonomic metrics

Requirements

• Snakemake 7.0+
• Conda or Mamba
• PacBio HiFi BAM files with corresponding barcode information

Installation

1. Clone this repository:

git clone https://github.com/yourusername/smk-bac-assembly.git
cd smk-bac-assembly

2. Install Snakemake (if not already installed):

conda create -n smk -c bioconda snakemake
conda activate smk

Configuration

Sample Setup

1. This workflow is prepared to use the CSV handed over by NGSP. Just copy it and store it as config/samples.csv:

BioSample,Barcode
sample_1,bc2170--bc2170
sample_2,bc2171--bc2171

Config File

Rename/copy the config file cp config/config_template.yaml config/config.yaml, then modify it to set:

• bam_dir: Path to your BAM files
• outdir: Output directory for results
• flye.genome_size: Approximate genome size for your organism
• Thread and memory allocations for each tool
• Location of the Bakta Database db_path

Usage

Run the complete workflow:

conda activate smk
cd workflow
snakemake --cores 32 --use-conda

Workflow Steps

1. Index BAM Files (index_bam)

• Creates samtools index (.bai) files required by NanoPlot
• Index files are marked as temporary and automatically cleaned up

2. Quality Control on Raw Reads (nanoplot_reads_qc)

• Generates comprehensive sequencing statistics with NanoPlot
• Creates visualizations of read quality and length distributions
• Outputs HTML report and tabular statistics

3. Prepare FASTQ (prepare_fastq)

• Converts BAM files to compressed FASTQ format
• Compressed files are temporary and cleaned after assembly

4. Genome Assembly (flye_assembly)

• Assembles long reads using Flye de novo assembler
• Produces assembly FASTA, assembly graph (GFA), and statistics

5. Assembly QC (assembly_qc)

• Extracts quality metrics from assembled genomes
• Uses seqkit to calculate N50, contig count, GC content, etc.

6. Summary Report (assembly_qc_summary)

• Combines QC metrics from all samples into a single summary table
• Facilitates comparison across all samples

7. Completeness Assessment (busco_run)

• Evaluates assembly completeness using BUSCO against conserved bacterial orthologs
• Provides completeness scores and missing genes analysis

8. Taxonomic Validation (pgap_taxcheck)

• Performs taxonomic classification using PGAP
• Validates assembly taxonomy with ANI-based methods
• Generates detailed taxonomic reports

9. Summary Tables (nanoplot_qc_summary, busco_summary, taxcheck_summary)

• Creates consolidated QC summary from NanoPlot stats
• Generates BUSCO completeness summary across samples
• Generates taxonomic validation summary with top ANI hits

10. Genome Annotation (bakta_annot)

• Annotates assembled genomes using Bakta
• Identifies genes, CDS, tRNAs, tmRNAs, rRNAs, ncRNAs, and CRISPR arrays
• Identifies AMR genes with AMRFinderPlus
• Outputs annotation results in TSV format

11. AMR Summary (amr_summary)

• Extracts antimicrobial resistance genes from Bakta annotations
• Filters for NCBI Protein and NCBI Family entries
• Creates summary table with sample, contig, gene, and product information

Output Structure

results/
├── qc/
│   ├── sequencing/
│   │   └── nanoplot/
│   │       ├── report/
│   │       ├── stats/
│   │       ├── scatter/
│   │       └── nanoplot_qc_summary.tsv
│   └── assemblies/
│       ├── {sample}_assembly_stats.txt
│       └── assembly_qc_summary.txt
├── assemblies/
│   └── flye/
│       └── {sample}/
│           ├── assembly.fasta
│           ├── assembly_graph.gfa
│           └── assembly_info.txt
├── completeness/
│   └── busco/
│       ├── {sample}/
│       │   ├── full_table.tsv
│       │   ├── missing_busco_list.tsv
│       │   └── short_summary.txt
│       └── busco_summary_{lineage}.tsv
├── annotation/
│   ├── taxcheck/
│   │   ├── {sample}.ani-tax-report.txt
│   │   └── taxcheck_summary.tsv
│   └── bakta/
│       └── {sample}/
│           ├── {sample}.tsv
│           └── {sample}_amr_summary.tsv
├── benchmarks/
└── logs/

Testing

Test dataset in test_data/:

• PacBio HiFi 100 reads unaligned BAM files
• 2 Samples 77SG_Aer_FM and 137DG-Aer_FM

Documentation

• Contributing Guidelines
• Code of Conduct

Tools Used

• NanoPlot - Long-read QC and visualization
• Flye - Long-read de novo assembly
• Seqkit - Sequence toolkit for statistics
• BUSCO - Benchmarking Universal Single-Copy Orthologs for completeness assessment
• PGAP - Prokaryotic Genome Annotation Pipeline for taxonomic validation
• Bakta - Rapid and standardized annotation of bacterial genomes
• Samtools - BAM file manipulation

Citation

If you use this workflow, please cite the respective tools:

• Flye: Kolmogorov M, et al. Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol. 2019.
• NanoPlot: De Coster W, et al. NanoPlot: visualizing and correcting long reads. Bioinformatics. 2017.
• Seqkit: Shen W, et al. SeqKit: a cross-platform and ultrafast toolkit for FASTA/Q file manipulation. PLoS ONE. 2016.
• BUSCO: Seppey M, et al. BUSCO: assessing genome assembly and annotation completeness. Methods Mol Biol. 2019.
• PGAP: Tatusova T, et al. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res. 2016.
• Bakta: Schwengers O, et al. Bakta: rapid and standardized annotation of bacterial genomes via alignment-free sequence identification. Microb Genom. 2021.

License

This project is licensed under the MIT License - see LICENSE.md for details.

Support

For issues, questions, or contributions, please use the GitHub Issues page or submit a pull request.

Acknowledgments

This workflow follows the WorkflowHub best practices for reproducible bioinformatics workflows.