RP-REP

RP-REP processes ribosomal profiling and RNA-Seq FASTQ data to quantify gene-level translation and report translational dynamics for biomarker discovery and differential translation analysis.

Key Features:

• Data Handling: Processes FASTQ files from local storage, AWS S3, or the Sequence Read Archive (SRA).
• Contaminant Filtering: Filters contaminant RNA sequences to reduce non-ribosomal signal.
• Footprint Enrichment: Enriches true ribosomal footprints to focus analysis on translating fragments.
• Reference Alignment and Quantification: Aligns reads to reference sequences and quantifies gene-level translation.
• Gene Body Coverage: Performs gene body coverage analysis to assess translation across gene regions.
• CRAM Compression: Compresses aligned sequencing data into CRAM format for storage efficiency.
• Alignment Quality Control: Executes reference alignment QC steps to assess alignment quality.
• Normalization and Visualization: Applies data normalization and multivariate data visualization methods.
• Differential Translation: Identifies differentially translated genes between conditions.
• Advanced Visualization: Generates heatmaps, co-translated gene clusters, and enriched pathway visualizations.
• Comparative Analysis: Compares RNA-Seq and ribosomal profiling results to assess translational efficiency per gene.
• Output Generation: Produces a comprehensive PDF report and publication-ready table and figure files.

Scientific Applications:

• Biomarker Discovery: Supports genome-wide characterization of protein synthesis for biomarker identification and detection of treatment-responsive genes.
• Translational Dynamics: Enables analysis of translational dynamics across cellular compartments and time courses, including cytosol and endoplasmic reticulum fractions.
• Infection Studies: Facilitates investigation of viral infection effects on host translation, exemplified by dengue virus infection time-course analysis in human Huh7 cells (6 h, 12 h, 24 h, 40 h post-infection).
• Pathway and Cluster Analysis: Identifies enriched pathways and co-translated gene clusters to interpret regulatory changes in translation.

Methodology:

Processes FASTQ inputs (local/S3/SRA); filters contaminant RNA; enriches ribosomal footprints; performs reference alignment and gene translation quantification; conducts gene body coverage analysis and alignment QC; compresses alignments to CRAM; applies data normalization and multivariate visualization; identifies differentially translated genes; generates heatmaps, co-translated gene clusters, and pathway enrichment outputs; compares RNA-Seq and ribosomal profiling for translational efficiency and produces PDF, table, and figure outputs.