🧞‍♂️ GeneGenie: RNA-seq Preprocessing Pipeline for Mycobacterium tuberculosis

GeneGenie is a modular, containerized workflow for preprocessing Mycobacterium tuberculosis RNA-seq data.

It supports both single- and paired-end sequencing formats and performs quality control, alignment, quantification, and reporting.

The pipeline is implemented in Nextflow DSL2, ensuring reproducibility and scalability.

---

✨ Key Features

• Input Validation: Uses seqkit for FASTQ validation; invalid samples are excluded and reported.
• Quality Control & Trimming: Supports trimgalore and fastp for adapter and quality trimming.
• Alignment: Offers bowtie2 and STAR aligners, with automatic index generation.
• BAM Processing: SAM to BAM conversion, sorting using samtools
• Quantification: Supports both featureCounts and HTSeq for gene-level quantification.
• Comprehensive Reporting: Aggregates QC and summary metrics via MultiQC.
• Containerized Execution: The pipeline runs in a dedicated Singularity container for reproducibility.
• Parameter Profiles: Predefined profiles for possible tool combinations.

---

🏃‍♂️ Usage

Project directory structure:

GeneGenie-1.0/
  ├── containers/
  ├── modules/
  ├── output/
  ├── reference/                     
  │   ├── data
  │   ├── genome.gtf                 
  │   └── genome.fasta               
  ├── workflows
  │   └── rnaseq.nf                 
  ├── genegenie.nf                                    
  ├── multiqc_config.yaml             
  ├── nextflow.config
  ├── nextflowrun.sh
  └── README.md
                    

Downloading containers

This pipeline uses the following Singularity containers from BioContainers:

• bowtie2:2.5.2--12e15c204b09f691 - Read alignment
• star:2.7.11a--0f5e3d475719bcac - Read alignment
• htseq:2.0.3--3205f67d4c550865 - Read counting
• fastp:0.23.4--b69359f46d2a8ebf - Read Quality control and trimming
• trim-galore:0.6.10--bc38c9238980c80e - Quality control and adapter trimming
• samtools:1.19.2--fbfb56ef5299fcef - SAM/BAM processing
• picard:3.4.0--2976616e7cbd4840 - BAM processing
• subread:2.0.6--2dd2dd526de026fd - Feature counting
• seqkit:2.10.0--9a5d37887d7c4e09 - Sequence toolkit (Fastq validation)
• multiqc:1.21--d44678e7b9933bf6 - Reporting

Example of command to download star container:

singularity pull oras://community.wave.seqera.io/library/star:2.7.11a--0f5e3d475719bcac

Running with a Profile

GeneGenie supports several profiles for six tool combinations. Use the -profile flag to select a profile:

Profile	QC Tool	Aligner	Quantification
TBF	trimgalore	bowtie2	featurecounts
TBH	trimgalore	bowtie2	htseq
FSF	fastp	star	featurecounts
FSH	fastp	star	htseq
TSH	trimgalore	star	htseq
TSF	trimgalore	star	featurecounts

Example use:

nextflow run GeneGenie.nf -profile TBF

Main parameters

• --input: Path to input CSV file (required)
• --read_type: single or paired (default: paired)
• --outdir: Output directory (default: ${projectDir}/output)
• --genome_fasta: Reference genome FASTA file (required)
• --gtf: GTF annotation file (required for quantification)
• --qc_tool: trimgalore or fastp
• --aligner: bowtie2 or star
• --quantification: featurecounts or htseq

You may override parameters at runtime like so:

nextflow run genegenie.nf -profile TSF \
  --input /path/to/reference/samplesheet.csv \
  --outdir /path/to/output \
  --gtf /path/to/reference/genomic.gtf \
  --genome_fasta /path/to/reference/genomic.fna \
  --max_cpus 8 \
  --max_memory 16.GB \
  --max_time 48.h \
  --read_type single

🛠️ Workflow Steps

Step	Tool(s)	Output Directory
Input Validation	seqkit	seqkit/
QC & Trimming	trimgalore / fastp	trimgalore/ or fastp/
Alignment	bowtie2 / STAR	bowtie2/ or star/
BAM Processing	samtools	samtools/
Quantification	featureCounts / HTSeq	featurecounts/ or htseq/
Reporting	MultiQC	multiqc/

All output files are organized under the specified --outdir.

---

📦 Output Files

• seqkit/validation_results.txt: Per-sample validation status.
• trimgalore/, fastp/: Cleaned FASTQ and QC logs.
• bowtie2/, star/: Alignment files (SAM/BAM).
• samtools/: Sorted BAMs, alignment metrics.
• featurecounts/, htseq/: Gene count tables.
• multiqc_report.html: Aggregated summary report.
• pipeline_info/: Execution reports, timeline, trace, and DAG.

---

⚡ Requirements

• Nextflow 21.04.0 or higher
• Apptainer/Singularity
• Sufficient disk space for intermediate and output files

---

🧠 Note

• Invalid samples are excluded after validation and reported in the output.

---

GeneGenie

🧞‍♂️ Reproducible, containerized, and modular workflow for M. tuberculosis RNA-seq preprocessing

---