MPRAdecoder

MPRAdecoder processes raw massively parallel reporter assay (MPRA) data to identify barcode (BC)–region of interest (ROI) associations, quantify BC expression from RT-PCR and next-generation sequencing (NGS) relative to plasmid DNA, and compute ROI-level activities.

Key Features:

• BC–ROI identification: Identifies unambiguous (genuine) internal barcodes (BCs) and their associated regions of interest (ROIs) from raw MPRA/mapping NGS samples.
• Ambiguity filtering: Detects and excludes ambiguous BCs, ambiguous BC–ROI associations, and ambiguous mutant variants from downstream analysis.
• Normalization of BC expression: Calculates normalized expression levels for each BC using transcript-level BC counts (RT-PCR) with normalization relative to plasmid DNA abundance measured by NGS.
• ROI-level aggregation: Computes averaged activity values for each ROI from associated BC measurements.
• Raw NGS processing: Processes raw MPRA and mapping NGS data to recover sequences and associations between BCs and ROIs.
• Graphical visualization: Produces graphical visualizations of processed MPRA data for downstream interpretation.

Scientific Applications:

• Functional screening of regulatory elements: Evaluates the regulatory potential of DNA regulatory elements and their mutant variants using MPRA-derived expression measurements.
• Decoding unknown libraries: Recovers BC and ROI sequences and their associations when sequence identities and mappings are not known a priori.
• Quantitative comparison of regulatory activity: Enables quantitative comparison of ROI activities by using normalized BC expression and averaged ROI values.

Methodology:

Processes raw MPRA and mapping NGS reads, identifies genuine BCs and associated ROIs, excludes ambiguous cases, calculates normalized BC expression relative to plasmid DNA, computes averaged ROI values, and generates graphical visualizations.