Nessie: A de novo genome assembly workflow for mammalian species using long reads

Introduction

This workflow supports sequecing data from both Oxford Nanopore and Pacbio HiFi sequencers, built with snakemake for maximum compatibility. The pipeline is based on the one used in De Novo Genome Assembly for an Endangered Lemur Using Portable Nanopore Sequencing in Rural Madagascar(Hauff et. all, 2025).

Workflow

Pipeline structure

Project structure

.
├── config
│   └── config.yaml
├── data
├── docs
├── benchmarks
├── kraken2_db.sh
├── LICENSE
├── logs
├── README.md
├── results
├── rules
│   ├── assembly.smk
│   ├── custom_k2_db.smk
│   ├── decontamination.smk
│   ├── envs
│   │   ├── assembly.yaml
│   │   ├── decontamination.yaml
│   │   ├── polish.yaml
│   │   ├── qc.yaml
│   │   ├── rm_haplotigs.yaml
│   │   └── trim_adapters.yaml
│   ├── polish.smk
│   ├── qc.smk
│   ├── rm_haplotigs.smk
│   └── trim_adapters.smk
├── scripts
│   └── reset.sh
├── setup.sh
├── snakefile
└── workflow.sh

System requirements

The workflow works mainly on Linux x86-64 HPC systems. It's currently being tested on a macOS system with M4 ARM CPU.

The total resources needed are based on the size of the data and the genome that is been analyzed.

Recomended specs

• GNU Linux 64 bit
• x86-64 CPU Architecture, Min: 32 Cores
• A lot of RAM, Min: 120GB
• Slurm Workload Manager

Some recomended options on threads: (These are the default settings in config.yaml based on the Saccharomyces cerevisiae)

Tool	Threads
samtools	16
porechop	16
flye	32
hifiasm	32
medaka	8
purge_haplotigs	16
kraken2	16
nanostat	8
quast	8
busco	16
multiqc	8

Depedencies

• Flye
• Hifiasm
• Porechop
• Medaka
• Purge_haplotigs
• Nanostat
• QUAST
• BUSCO
• multiQC
• Kraken 2
• Seqkit
• Samtools
• NCBI Datasets (Optional)

Installation

1. Conda

In order to run the workflow, conda must be installed. Bellow are the full steps for installing and setup conda and bioconda for Linux machines. If you want to experiment with other configurations and distros, here are the instructions.

Taken from the official conda documentation.

Download the installer:

• Miniconda installer for Linux --> link
• Anaconda Distribution installer for Linux --> link
• Miniforge installer for Linux --> link

Verify your installer hashes --> link

Run this command:

bash <conda-installer-name>-latest-Linux-x86_64.sh

conda-installer-name will be one of "Miniconda3", "Anaconda", or "Miniforge3".

Then follow instructions on screen.

Verify installation with:

conda list

Setup Bioconda:

To add the bioconda channel on ~/.condarc file, run the following in the correct order:

conda config --add channels bioconda
conda config --add channels conda-forge
conda config --set channel_priority strict

Even if you have a previous bioconda setup, it is recommended to re-run these commands.

2. Setup workflow

Download the repository: You can simply git clone the repo, or download it manually from GitHub or the new GitHub CLI app.

git clone https://github.com/mikeph52/nessie.git

Run setup.sh from inside the folder:

./setup.sh
# or
bash setup.sh

Follow instructions. The setup.sh script enables you to rename the folder to your liking, creates importand project folders, checks for an existent conda installation and installs snakemake if it's not installed.

Here's an example of the script:

-------------------------------------
|              Nessie               |
|De novo assembly snakemake workflow|
|         by mikeph52, 2026         |
-------------------------------------
Enter project name: pseudomonas_syringae
The following changes will happen:
 - Name project pseudomonas_syringae
 - Create directories: data/ logs/ results/
 - Create subfolders on: data/ results/
 - Check conda availability
 - Install snakemake if it is not installed
 - Move setup.sh to scripts/
Continue? (Y/N): 

3. Reset installation

If you want to revert changes made by the setup.sh script, run the reset.sh inside the scripts/ folder.

Usage

Workflow configuration

All settings and configuration is managed from the config.yaml, found in the config/ folder. Inside the config file there are 5 sections:

• Samples Load your .BAM files here.

samples: #.BAM files
  - sample1
  - sample2
  # etc

• Assembler

Select the assembler to use on the assembly between Flye for Nanopore reads and Hifiasm for PacBio HiFi reads.

assembler: flye #flye or hifiasm

• Genome size

Enter the genome size of the organism. It's required for Flye and QUAST to run.

genome_size: "500m"

• Threads

Select the threads for each tool, depending on the available resources and the

threads:
  samtools: 16
  porechop: 16
  flye: 32
# . . . 

• Tools arguments Here you can add additional arguments for the tools in this workflow.

flye:
  read_type: "--nano-hq"    # --nano-raw # --nano-hq # --pacbio-raw # --pacbio-hifi
  extra_args: ""

busco:
  lineage: "fungi_odb10" # example for S.cerevisiae
  mode: "genome"
  extra_args: ""
# . . . 

Execution with Slurm

To execute the workflow in an HPC System with the Slurm workflow manager installed, execute the workflow.sh script.

sbatch workflow.sh

Manual execution

Inside the project folder, run the following:

snakemake -j 20 # select the cores you need

Changelog

Changelog starts from the first public version (v.0.18.1, 1/6/2026)

Version 0.20.1 (12/6/2026)

• Command benchmark: added to every rule.smk to capture resources.
• Banner logo added.
• Success and error warning message added.
• correct multiQC formatting issues.
• Minor bugs fixed.

Version 0.19.1 (7/6/2026)

• Fix issue (#8)

Version 0.18.1 (1/6/2026)

• Old rm_haplotigs.smk removed.
• Fixed automatic coverage cutoff computation.
• Formatting issues fixed.
• Fixed medaka version in conda env.

Tests & Benchmarks

The workflow has been tested on 3 assemblies so far:

• Clonostachys chloroleuca strain Cc878
• Eulemur rufifrons
• Alexandromys oeconomus

For the results of each test run, visit benchmarks.

Acknowledgements

All data used for the development of this workflow were provided by the Institute of Marine Biology, Biotechnology and Aquaculture (IMBBC) of the Hellenic Centre for Marine Research (HCMR), Heraklion, Crete. This workflow was developed and executed on the Zorbas HPC infrastructure of IMBBC-HCMR (Zafeiropoulos et al., 2021).

References

• Hauff, L., Rasoanaivo, N.E., Razafindrakoto, A., Ravelonjanahary, H., Wright, P.C., Rakotoarivony, R. and Bergey, C.M. (2025), De Novo Genome Assembly for an Endangered Lemur Using Portable Nanopore Sequencing in Rural Madagascar. Ecol Evol, 15: e70734. https://doi.org/10.1002/ece3.70734

• Kolmogorov, M., Yuan, J., Lin, Y. et al. Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol 37, 540–546 (2019). https://doi.org/10.1038/s41587-019-0072-8

• Cheng, H., Asri, M., Lucas, J., Koren, S., Li, H. (2024) Scalable telomere-to-telomere assembly for diploid and polyploid genomes with double graph. Nat Methods, 21:967-970. https://doi.org/10.1038/s41592-024-02269-8

• Tegenfeldt F., Kuznetsov D., Manni M., Berkeley M., Zdobnov E.M., Kriventseva E.V. OrthoDB and BUSCO update: annotation of orthologs with wider sampling of genomes. Nucleic Acids Research, Volume 53, Issue D1, 6 January 2025, Pages D516–D522, https://doi.org/10.1093/nar/gkae987

• Alla Mikheenko, Vladislav Saveliev, Pascal Hirsch, Alexey Gurevich, WebQUAST: online evaluation of genome assemblies, Nucleic Acids Research (2023) 51 (W1): W601–W606. doi: 10.1093/nar/gkad406 First published online: May 17, 2023

• Wood, D.E., Lu, J. & Langmead, B. Improved metagenomic analysis with Kraken 2. Genome Biol 20, 257 (2019). https://doi.org/10.1186/s13059-019-1891-0

• Shen, Wei, BotondSipos, and LiuyangZhao. 2024. “SeqKit2: A Swiss Army Knife for Sequence and Alignment Processing.” iMeta3, e191. https://doi.org/10.1002/imt2.191

• Roach, M.J., Schmidt, S.A. & Borneman, A.R. Purge Haplotigs: allelic contig reassignment for third-gen diploid genome assemblies. BMC Bioinformatics 19, 460 (2018). https://doi.org/10.1186/s12859-018-2485-7

• Wick RR, Judd LM, Gorrie CL, Holt KE. Completing bacterial genome assemblies with multiplex MinION sequencing. Microb Genom. 2017;3(10):e000132. Published 2017 Sep 14. doi:10.1099/mgen.0.000132

• Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO, Whitwham A, Keane T, McCarthy SA, Davies RM, Li H, Twelve years of SAMtools and BCFtools, GigaScience (2021) 10(2) giab008 [33590861]

• Philip Ewels, Måns Magnusson, Sverker Lundin, Max Käller, MultiQC: summarize analysis results for multiple tools and samples in a single report, Bioinformatics, Volume 32, Issue 19, October 2016, Pages 3047–3048, https://doi.org/10.1093/bioinformatics/btw354

• O’Leary NA, Cox E, Holmes JB, Anderson WR, Falk R, Hem V, Tsuchiya MTN, Schuler GD, Zhang X, Torcivia J, Ketter A, Breen L, Cothran J, Bajwa H, Tinne J, Meric PA, Hlavina W, Schneider VA. Exploring and retrieving sequence and metadata for species across the tree of life with NCBI Datasets. Sci Data. 2024 Jul 5;11(1):732. doi: 10.1038/s41597-024-03571-y. PMID: 38969627; PMCID: PMC11226681.

• Wouter De Coster, Svenn D’Hert, Darrin T Schultz, Marc Cruts, Christine Van Broeckhoven, NanoPack: visualizing and processing long-read sequencing data, Bioinformatics, Volume 34, Issue 15, August 2018, Pages 2666–2669, https://doi.org/10.1093/bioinformatics/bty149

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