mkrg01/genome_assembly_pipeline

An integrated pipeline for eukaryotic genome assembly and gene annotation

Overview

Latest release: None, Last update: 2026-06-14

Share link: https://snakemake.github.io/snakemake-workflow-catalog?wf=mkrg01/genome_assembly_pipeline

Quality control: linting: passed formatting: failed

Deployment

Step 1: Install Snakemake and Snakedeploy

Snakemake and Snakedeploy are best installed via the Conda package manager. It is recommended to install conda via Miniforge. Run

conda create -c conda-forge -c bioconda -c nodefaults --name snakemake snakemake snakedeploy

to install both Snakemake and Snakedeploy in an isolated environment. For all following commands ensure that this environment is activated via

conda activate snakemake

For other installation methods, refer to the Snakemake and Snakedeploy documentation.

Step 2: Deploy workflow

With Snakemake and Snakedeploy installed, the workflow can be deployed as follows. First, create an appropriate project working directory on your system and enter it:

mkdir -p path/to/project-workdir
cd path/to/project-workdir

In all following steps, we will assume that you are inside of that directory. Then run

snakedeploy deploy-workflow https://github.com/mkrg01/genome_assembly_pipeline . --tag None

Snakedeploy will create two folders, workflow and config. The former contains the deployment of the chosen workflow as a Snakemake module, the latter contains configuration files which will be modified in the next step in order to configure the workflow to your needs.

Step 3: Configure workflow

To configure the workflow, adapt config/config.yml to your needs following the instructions below.

Step 4: Run workflow

The deployment method is controlled using the --software-deployment-method (short --sdm) argument.

To run the workflow using a combination of conda and apptainer/singularity for software deployment, use

snakemake --cores all --sdm conda apptainer

Snakemake will automatically detect the main Snakefile in the workflow subfolder and execute the workflow module that has been defined by the deployment in step 2.

For further options such as cluster and cloud execution, see the docs.

Step 5: Generate report

After finalizing your data analysis, you can automatically generate an interactive visual HTML report for inspection of results together with parameters and code inside of the browser using

snakemake --report report.zip

Configuration

The following section is imported from the workflow’s config/README.md.

Configuration Guide

This document explains:

  1. Required input files to be placed in the raw_data directory.

  2. Configuration parameters to set in config/config.yml.

1. Input Files (raw_data/)

The full assembly workflow requires PacBio HiFi reads. The external-assembly annotation workflow (workflow/Snakefile.annotation) instead requires an assembly FASTA via external_assembly. Paired-end RNA-seq reads are required when running targets that include gene prediction (gene_prediction_all, circos_plot_all, or all).

Place your raw sequencing files in the raw_data directory with the following naming conventions:

File Type

Naming Pattern

Example

PacBio HiFi reads

*.hifi_reads.bam

SAMPLE1.hifi_reads.bam

Index for HiFi reads

*.hifi_reads.bam.pbi

SAMPLE1.hifi_reads.bam.pbi

External assembly FASTA (annotation workflow only)

Any path set in external_assembly

raw_data/GCA_054852875.1_ASM5485287v1_genomic.fna

Ultra-long ONT reads (optional)

Any FASTQ path in config (.fastq.gz, .fq.gz, .fastq, .fq)

raw_data/ont_reads.fastq.gz

Hi-C reads R1 (optional)

One or more FASTQ paths in hic_reads_r1 (.fastq.gz, .fq.gz, .fastq, .fq)

raw_data/hic_R1.fastq.gz

Hi-C reads R2 (optional)

One or more FASTQ paths in hic_reads_r2 (.fastq.gz, .fq.gz, .fastq, .fq)

raw_data/hic_R2.fastq.gz

Paired-end RNA-seq (R1)

*_1.fastq.gz, *_1.fq.gz, *_1.fastq, or *_1.fq

RNASEQ1_1.fastq.gz

Paired-end RNA-seq (R2)

*_2.fastq.gz, *_2.fq.gz, *_2.fastq, or *_2.fq

RNASEQ1_2.fastq.gz

Notes:

  • The pipeline will automatically detect and process multiple PacBio HiFi and RNA-seq samples, if present.

  • PacBio HiFi reads are not required when running the external-assembly annotation workflow with workflow/Snakefile.annotation.

  • RNA-seq raw FASTQ inputs can be gzipped or plain text. The pipeline normalizes them during preprocessing, so downstream rules keep using the same internal filenames.

  • Keep only one raw RNA-seq file per sample and read pair. For example, do not place both SAMPLE_1.fastq.gz and SAMPLE_1.fq in raw_data/.

  • Ultra-long ONT reads are optional and can improve assembly quality when integrated with HiFi reads. See the hifiasm documentation for details.

  • Hi-C reads are optional and should be provided as matching R1/R2 paths in hic_reads_r1 and hic_reads_r2. Multiple lanes can be listed in matching order. If Hi-C reads are configured, the pipeline uses them for hifiasm phasing, runs YaHS scaffolding on each assembly listed in selected_assemblies, and prepares Juicebox-ready contact maps. See the hifiasm documentation and the YaHS documentation for details.

  • LongStitch is enabled by default. It uses the curated HiFi reads produced by this workflow.

2. Configuration File (config/config.yml)

Edit config/config.yml to match your dataset and analysis requirements.
The sections below follow the order of config/config.yml.

Runtime

Parameter

Description

Example

pipeline_version

Container image tag used by the entrypoint Snakefiles. The workflow builds the full image URI as docker://aurelia01/genome_assembly_pipeline:<pipeline_version>.

"v0.6.15"

organism_name

Filesystem-safe organism label used for output file prefixes and GenBank source/metadata. Use underscores instead of spaces; legacy assembly_name is still accepted as a fallback.

"Dioncophyllum_thollonii"

genome_version

Filesystem-safe version label for this genome release. Used for release directories and output file names; legacy assembly_version is still accepted as a fallback.

"v1.0"

external_assembly

Required only with workflow/Snakefile.annotation. Set this to a single FASTA path string. Gzipped FASTA inputs are decompressed when staged into results/external/assembly/.

"raw_data/GCA_054852875.1_ASM5485287v1_genomic.fna"

Hifiasm Genome Assembly

Parameter

Description

Example

selected_assemblies

One or more assemblies to process after hifiasm. Allowed values: {"primary", "hap1", "hap2"}. Downstream outputs are written under assembly-specific subdirectories such as results/fcs/assembly/<selected_assembly>/... or results/release/<genome_version>/nuclear/<selected_assembly>/.... Release outputs are produced for every selected assembly. When Hi-C integration is enabled, these correspond to hifiasm hic.p_ctg, hic.hap1.p_ctg, and hic.hap2.p_ctg outputs, which are then scaffolded with YaHS, exported as Juicebox-ready contact-map files under results/juicebox/<selected_assembly>/..., and written under results/yahs/assembly/<selected_assembly>/... before RepeatMasker and later steps. Organelle release assets are staged separately under results/release/<genome_version>/organelle/... according to oatk_organelle and organelle_annotation.

["primary", "hap1", "hap2"]

hifiasm_dual_scaf

Optional: Enable hifiasm self-scaffolding by adding --dual-scaf. {true, false}

false

ont_reads

Optional: Path to ultra-long ONT reads in FASTQ format (.fastq.gz, .fq.gz, .fastq, or .fq). Set to null to disable ONT integration. See hifiasm docs

null

hic_reads_r1

Optional: Hi-C read 1 input for hifiasm phasing, YaHS scaffolding, and Juicebox-ready contact maps. Set to null to disable Hi-C integration, or provide one or more FASTQ paths as a YAML list. Must be paired with hic_reads_r2. See hifiasm docs

["raw_data/hic_R1.fastq.gz"]

hic_reads_r2

Optional: Hi-C read 2 input for hifiasm phasing, YaHS scaffolding, and Juicebox-ready contact maps. Must match hic_reads_r1 in length and order.

["raw_data/hic_R2.fastq.gz"]

Oatk Organelle Assembly

Parameter

Description

Example

oatk_lineage

Lineage of the Oatk HMM profile database. Lineage list

"magnoliopsida"

oatk_organelle

Organelle to assemble. {"mitochondrion", "chloroplast", "mitochondrion_and_chloroplast"}. Legacy aliases mito, pltd, and mito_and_pltd are still accepted.

"mitochondrion_and_chloroplast"

oatk_minimum_kmer_coverage

Minimum kmer coverage used for Oatk. Use "auto" to estimate the value as 7 times the nuclear HiFi read coverage inferred from total HiFi read bases and the first selected hifiasm assembly length, or set a positive integer to override. The resolved value and metadata are written under results/oatk/auto/. Instructions

"auto"

Contamination Screening

Parameter

Description

Example

taxid

NCBI Taxonomy ID for the target organism. Used by FCS-GX screening and organelle GenBank source db_xref. Legacy fcs_gx_taxid is still accepted as a fallback. NCBI Taxonomy Tree

"122299" for Dioncophyllum thollonii

LongStitch Misassembly Correction and Scaffolding

Parameter

Description

Example

longstitch_enabled

Run LongStitch on the FCS-cleaned assembly before YaHS and downstream analysis. LongStitch uses the curated HiFi reads produced by this workflow. When enabled, the haploid genome size passed as G is derived from the FCS-cleaned assembly sum_len in the workflow-generated SeqKit stats table. Set to false to skip this step. {true, false}

true

YaHS Scaffolding

Parameter

Description

Example

yahs_restriction_enzymes

Optional: Restriction enzyme motif(s) passed to YaHS with -e during Hi-C scaffolding after FCS cleanup and optional LongStitch correction. Set to null to use YaHS defaults. This parameter is used only when both hic_reads_r1 and hic_reads_r2 are set. See YaHS docs

"GATC"

Quality Assessment

Parameter

Description

Example

busco_lineage_dataset

BUSCO lineage dataset for genome completeness assessment. Lineage list

"embryophyta_odb12"

tidk_clade

Clade for tidk find. Lineage list

"Caryophyllales"

tidk_telomeric_repeat_unit

A telomeric repeat unit for tidk search. A Telomeric Repeat Database

"AAACCCT"

Softmasking

Parameter

Description

Example

dfam_version

Version of the Dfam database for RepeatMasker. Dfam releases

"3.9"

dfam_partitions

Dfam partitions. See README.txt.

"0,5,6" (Viridiplantae)

dfam_lineage_name

Name of the Dfam lineage to use.

"Viridiplantae"

Gene Prediction

Parameter

Description

Example

orthodb_version

Version of the OrthoDB database (used by Braker3). ProtHint instructions

"12"

orthodb_lineage

OrthoDB lineage dataset to use. Lineage list

"Viridiplantae"

orthodb_md5sum

MD5 checksum of the OrthoDB database. Checksums

"34c1f027a1a7b10f225b69fbd5500587"

Visualization

Parameter

Description

Example

min_long_contig_length

Minimum contig length to be considered a “long contig” for downstream visualization (e.g., Circos plots).

1_000_000

circos_plot_tracks

Track configuration for the Circos plot

{id: "gene", label: "Gene model", color: "#4C72B0", window_size: 500_000}

circos_plot_x_major_tick_interval

Major tick interval (in bp) for the x-axis in Circos plots. Major ticks are labeled.

10_000_000

circos_plot_x_minor_tick_interval

Minor tick interval (in bp) for the x-axis in Circos plots. Minor ticks are unlabeled.

5_000_000

Organelle Annotation

Parameter

Description

Example

organelle_annotation

Annotation tools for Oatk-assembled organelle genomes. Mitochondrion: {"pmga", null}. Chloroplast: {"pga_v2", null}. Omitted keys, per-organelle null, or top-level organelle_annotation: null skip annotation. Legacy keys mito and pltd are still accepted. Oatk organelle FASTA record IDs are prefixed before annotation/submission (mt_ for mitochondrion, cp_ for chloroplast) so GenBank LOCUS names and submission FASTA headers stay unique and consistent. For PMGA/PGA, circular single-record inputs are first annotated once to choose an internal feature-free origin, then rotated before final annotation so origin-spanning genes are less likely to be missed; the rotated annotation FASTA is used for RNA-editing evidence mapping and submission. PMGA/PGA GenBank outputs are then post-curated with RNA-seq and HiFi evidence to add CDS /translation and accepted /exception="RNA editing" qualifiers. Normal RNA-editing calls use accept thresholds of RNA depth >= 10, edited reads >= 3, edit fraction >= 0.10, base quality >= 30, mapping quality >= 30, DNA/HiFi depth >= 10, and DNA/HiFi alternate fraction <= 0.10; CDS-essential rescue sites that restore a valid start codon, terminal stop codon, or premature-stop rescue may use a separate one-read rescue threshold with DNA/HiFi alternate fraction <= 0.10. Sites with DNA/HiFi alternate support above the threshold are retained as likely_genomic_variant evidence and summarized for genome sequence or variant review. PMGA and PGA v2.0 are downloaded automatically only when selected.

{mitochondrion: "pmga", chloroplast: "pga_v2"}

organelle_reference_cds_qc

Optional reference CDS/protein QC for PMGA/PGA annotations. Set an organelle reference_dir to null to skip reference QC and reference-inferred RNA-editing rescue for that organelle. When reference_dir is set, the pipeline emits pre/post RNA-editing QC tables and conservatively tries closely related reference CDS/protein evidence for residual invalid CDS after RNA-seq curation. fix_hifi_frameshifts: true is allowed only for chloroplast/PGA and may correct well-supported HiFi read-supported frameshift indels before rerunning PGA. Chloroplast/PGA requires a reference directory because PGA v2 itself is reference-based.

{chloroplast: {reference_dir: "plastid_reference", fix_hifi_frameshifts: false}, mitochondrion: {reference_dir: null, fix_hifi_frameshifts: false}}

Choosing Organelle Reference CDS QC References

When organelle_annotation.chloroplast is set to pga_v2, place one or a few plastid GenBank files (.gb, .gbk, or .gbff) in organelle_reference_cds_qc.chloroplast.reference_dir. PGA v2 uses these annotated references to transfer chloroplast/plastid gene annotations, so the reference choice can affect annotation completeness and naming. The same references are also used for chloroplast CDS/protein QC and reference-inferred RNA-editing rescue.

For PMGA/mitochondrion, organelle_reference_cds_qc.mitochondrion.reference_dir is optional. If provided, place one or a few mitochondrial GenBank files (.gb, .gbk, or .gbff) in that directory. Those references are used for mitochondrial CDS/protein QC and reference-inferred RNA-editing rescue. Applied reference-inferred sites are recorded separately from RNA-seq-supported calls in the RNA-editing evidence sidecars and GenBank /inference qualifiers.

Useful NCBI Nucleotide searches for chloroplast/plastid references:

"<Species name>"[Organism] AND chloroplast[Title] AND "complete genome"[Title]
<Genus name>[Organism] AND chloroplast[Title] AND "complete genome"[Title]
<Family name>[Organism] AND chloroplast[Title] AND "complete genome"[Title]

Useful NCBI Nucleotide searches for mitochondrial references:

"<Species name>"[Organism] AND mitochondrion[Title] AND "complete genome"[Title]
<Genus name>[Organism] AND mitochondrion[Title] AND "complete genome"[Title]
<Family name>[Organism] AND mitochondrion[Title] AND "complete genome"[Title]

Prefer complete organelle GenBank records from the same species, genus, or family when available.

NCBI GenBank download examples:

mkdir -p plastid_reference
mkdir -p mitochondrial_reference

PLASTID_ACCESSION="NC_041245.1"
MITOCHONDRION_ACCESSION="<mitochondrion accession>"

curl -L "https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?db=nuccore&id=${PLASTID_ACCESSION}&rettype=gb&retmode=text" \
  -o "plastid_reference/${PLASTID_ACCESSION}.gbk"

curl -L "https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?db=nuccore&id=${MITOCHONDRION_ACCESSION}&rettype=gb&retmode=text" \
  -o "mitochondrial_reference/${MITOCHONDRION_ACCESSION}.gbk"

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