TranseqFlow-PE

Transcriptome upstream analysis pipeline with Nextflow for paired-end reads. It performs quality check with FASTQC and MultiQC, trimming with Trimmomatic, alignment with STAR, and quantification of RNA-seq data with FeatureCount, producing clean and reproducible results.

The Processs and the inegrated tools

FASTQC - fastqc: Performs quality control checks on raw sequencing data to generate quality reports.

MULTIQC - multiqc: Aggregates results from FASTQC into a single report for easier analysis.

TRIM - trimmpmaticPE: Uses Trimmomatic to remove low-quality bases and adapter sequences from paired-end reads, generating trimmed and cleaned reads.

INDEX - STAR: Creates a genome index using the reference transcriptome and annotation file with STAR to prepare for the alignment step.

MAPPING - STAR: Aligns the trimmed reads to the reference genome using STAR, producing SAM files for further processing.

BAM - samtools: Converts SAM files to BAM format, sorts and indexes them for downstream analysis using samtools.

FeatureCount - featureCounts: Quantifies gene expression by counting the number of reads aligned to each gene using featureCounts.

PS: you have to make sure you have installed NextFlow and all of these tools on your environment before using this pipline.

Input Parameters

The pipeline accepts the following input parameters:

reads: Path to paired-end reads in .fastq.gz format. The pipeline expects the reads to be organized as forward and reverse reads for each sample.

transcriptome_file: Path to the reference transcriptome file in FASTA format.

annotationfile_file: Path to the reference genome annotation file in GTF format.

outdir: Directory where the pipeline will store the output files.

Please make sure that your fastqs are in .fastq.gz format and all of reads are placed in raw_reads directory, and your reference and gtf files are stored in ref directory.

Usage

nextflow run transseqflowPE.nf --reads '/path/to/raw_reads/*_{1,2}.fastq.gz' --transcriptome_file '/path/to/ref/reference.fasta' --annotationfile_file '/path/to/ref/annotation.gtf'

Outputs

The results are saved in the specified output directory (outdir) and include:

• FASTQC quality reports

• A MultiQC summary report

• Trimmed reads

• BAM files (sorted and indexed)

• Gene count tables for each sample