Reproducible Bash workflows for chromosome-level genome assembly and functional annotation.
This repository releases exactly two Bash workflows:
| Script | Purpose |
|---|---|
genoflowHiFi_HiC_v0.1.0.sh |
PacBio HiFi assembly with Hi-C scaffolding, manual curation, polishing, evaluation, repeat annotation, and reporting |
functional_annotation_v2.sh |
RNA-seq processing, protein functional annotation, consensus tables, and descriptive expression and genome-annotation graphics |
Other development Bash workflows are not part of this public distribution.
functional_annotation_v2_R/ contains the three R helpers used by the public functional-annotation workflow. They are supporting source files, not additional Bash workflows.
genoflowHiFi_HiC_v0.1.0.sh implements the following stages:
PacBio HiFi reads + paired Hi-C reads
|
Hifiasm primary assembly
|
Optional FCS-Adaptor and FCS-GX decontamination
|
DeepVariant pre-polishing
|
Inspector correction
|
Chromap alignment and YaHS scaffolding
|
Required manual YaHS/JBAT curation
|
TGS-GapCloser2 gap filling
|
Final DeepVariant polishing and optional FCS-GX cleanup
|
QUAST, BUSCO, BlobToolKit, Qualimap, and MultiQC evaluation
|
RepeatModeler2, TEtrimmer, RepeatMasker, and Earl Grey
|
Final assembly and run summary
The primary assembly is written to:
07.final_asm/<Scientific_Name>.fasta
The workflow has a hard manual-curation boundary after YaHS Step 5.
Pass 1 creates the YaHS scaffolds and JBAT curation bundle:
working_dir="/path/to/project" \
pacbio_hifi="/path/to/hifi_reads.fastq.gz" \
pacbio_hifi_fasta="/path/to/hifi_reads.fasta.gz" \
hic_r1="/path/to/hic_R1.fastq.gz" \
hic_r2="/path/to/hic_R2.fastq.gz" \
TAXID="13604" \
BUSCO_LINEAGE="actinopterygii_odb12" \
run_hifiasm=TRUE \
run_fcs_early=TRUE \
run_deepvariant=TRUE \
run_inspector=TRUE \
run_yahs=TRUE \
run_tgsgapcloser2=FALSE \
run_final_asm=FALSE \
run_stats=FALSE \
run_repeats=FALSE \
bash genoflowHiFi_HiC_v0.1.0.shCurate the JBAT assembly and save the curated FASTA. The default handoff path is:
05.yahs/curated_assembly/<base>.curated.fa
Pass 2 resumes from that curated assembly:
working_dir="/path/to/project" \
pacbio_hifi="/path/to/hifi_reads.fastq.gz" \
pacbio_hifi_fasta="/path/to/hifi_reads.fasta.gz" \
hic_r1="/path/to/hic_R1.fastq.gz" \
hic_r2="/path/to/hic_R2.fastq.gz" \
curated_assembly="/path/to/project/05.yahs/curated_assembly/<base>.curated.fa" \
TAXID="13604" \
BUSCO_LINEAGE="actinopterygii_odb12" \
run_hifiasm=FALSE \
run_fcs_early=FALSE \
run_deepvariant=FALSE \
run_inspector=FALSE \
run_yahs=FALSE \
run_tgsgapcloser2=TRUE \
run_final_asm=TRUE \
run_stats=TRUE \
run_repeats=TRUE \
bash genoflowHiFi_HiC_v0.1.0.shImportant configuration variables are defined near the top of the script. Local executable, database, container, taxonomy, and input paths must be reviewed before execution.
01.hifiasm/ Primary assembly
02.fcs_decontam/ FCS screening and cleanup
03.deepvariant/ Pre-scaffold polishing
04.inspector/ Assembly correction
05.yahs/ Hi-C alignment, YaHS scaffolds, and curation handoff
06.tgsgapcloser2/ Gap-closing outputs
07.final_asm/ Final assembly
08.asm_stats/ Assembly evaluation
09.repeats/ Repeat libraries and annotations
10.summary/ Final run summaries
functional_annotation_v2.sh provides modular stages for:
- SRA download and FASTQ conversion.
fastpquality control and trimming.- HISAT2 alignment and indexed BAM generation.
- Gene-level counting with
Rsubread::featureCounts. - eggNOG-mapper annotation.
- InterProScan domain and protein-family annotation.
- DIAMOND searches against curated Swiss-Prot proteins.
- KofamScan KEGG Orthology assignment.
- Optional dbCAN CAZyme annotation.
- Consensus tables and descriptive RNA-seq and genome-annotation graphics.
The expression component supports descriptive fold-change plots for designs with one sample per organ. These outputs are not formal differential-expression tests because biological replication is absent.
RUN_MODE |
Behavior |
|---|---|
annotation_only |
Runs the five protein-annotation methods; this is the default |
all |
Enables all processing, annotation, and plotting stages |
plots_only |
Generates the R-based tables and plots from existing inputs |
preflight |
Validates all enabled inputs, databases, environments, and tools without running stages |
startup_check |
Exercises initialization with all stages disabled |
custom |
Uses only explicitly supplied RUN_* stage switches |
Basic invocation:
PROJECT_DIR="/path/to/project" \
EGAPX_OUT_DIR="/path/to/egapx/out" \
RUN_MODE="annotation_only" \
bash functional_annotation_v2.sh 8 4The two positional arguments are threads per job and parallel jobs. Every study-specific path and stage switch can be overridden with environment variables.
The default sample metadata contains the four SRA accessions used in the associated study. Supply 01_metadata/sra_metadata.tsv or set RUN_CREATE_METADATA=TRUE with an edited accession table for another study.
The following files must remain beside the Bash script under functional_annotation_v2_R/:
run_featurecounts_rsubread.R
rnaseq_single_sample_plots.R
egapx_genome_annotation_graphics_karyoploter.R
Alternatively, set R_SOURCE_DIR, FEATURECOUNTS_R_SOURCE, RNASEQ_R_SOURCE, and GENOME_GRAPHICS_R_SOURCE to external copies.
00_logs/ Logs and stage manifests
01_metadata/ SRA and sample metadata
02_sra/ SRA downloads
03_fastq/ Raw FASTQ files
04_fastp/ Trimmed reads and fastp reports
05_multiqc/ MultiQC reports
06_hisat2/ HISAT2 index, BAM files, and summaries
07_featurecounts/ Gene-count tables
08_eggnog_mapper/ eggNOG-mapper outputs
09_interproscan/ InterProScan outputs
10_diamond_annotation/ DIAMOND/Swiss-Prot outputs
11_kofamscan/ KofamScan outputs
12_dbcan/ dbCAN outputs
13_consensus_annotation/ Combined tables, scripts, and plots
Clone the repository and make the two public Bash workflows executable:
git clone https://github.com/bferrette/genoflowHiFi.git
cd genoflowHiFi
chmod 755 genoflowHiFi_HiC_v0.1.0.sh functional_annotation_v2.shThe workflows coordinate external software; they do not install it. Major assembly dependencies include Hifiasm, Chromap, YaHS, DeepVariant, Inspector, TGS-GapCloser2, QUAST, BUSCO, BlobToolKit, RepeatModeler2, TEtrimmer, RepeatMasker, Earl Grey, GNU Parallel, and Apptainer or Singularity.
Major functional-annotation dependencies include SRA Toolkit, fastp, HISAT2, SAMtools, GNU Parallel, MultiQC, eggNOG-mapper, InterProScan, DIAMOND, KofamScan, dbCAN, mamba, and R with the packages declared in R_REQUIRED_PACKAGES.
Exact versions, environment names, paths, databases, and optional stages are recorded in the scripts. Full production runs require substantial storage and compute resources.
The optional definitions under containers/ document BlobToolKit, GATK 4, and
TE-tools container builds. They do not install or build containers automatically.
Syntax validation:
bash -n genoflowHiFi_HiC_v0.1.0.sh
bash -n functional_annotation_v2.shStatic analysis, when ShellCheck is installed:
shellcheck -x genoflowHiFi_HiC_v0.1.0.sh
shellcheck -x functional_annotation_v2.shLow-impact initialization check for the annotation workflow:
project_dir="$(mktemp -d)"
PROJECT_DIR="${project_dir}" RUN_MODE=startup_check \
bash functional_annotation_v2.sh 1 1These checks do not replace validation on representative sequencing and annotation data.
The latest archived release is available through the Zenodo concept DOI. This DOI resolves to the most recent archived version.
For reproducible scientific citation, use the version-specific DOI associated with this release:
Version DOI: 10.5281/zenodo.21650438
Concept DOI: 10.5281/zenodo.21650437
Ferrette, B. L. S. (2026). genoflowHiFi: PacBio HiFi–Hi-C Genome Assembly and
Functional Annotation Workflows (version 0.1.0). Zenodo.
https://doi.org/10.5281/zenodo.21650438
The version DOI identifies the exact archived release used in an analysis. The concept DOI identifies the software project and resolves to its latest Zenodo version.
This software is distributed under the GNU General Public License v3.0. See LICENSE.