top of page

qPRO-sequencing Analysis

Quantitative Precision Run-On sequencing (qPRO-seq) captures nascent RNA transcription genome-wide at nucleotide resolution, providing a direct readout of RNA Polymerase II activity across the genome. Unlike steady-state RNA-seq—which measures accumulated transcript levels—qPRO-seq reveals active transcription as it happens, making it uniquely powerful for studying transcriptional pausing, elongation dynamics, enhancer RNA (eRNA) production, and rapid transcriptional responses to stimuli or perturbation.

​

As a technically demanding assay, qPRO-seq requires careful attention to spike-in normalisation, run-on efficiency, and the specialised bioinformatic approaches needed to accurately identify transcription units and promoter-proximal pause sites. At Porter-Turnbull Genomics Consultancy, we provide expert analysis of qPRO-seq data that goes beyond read alignment—delivering mechanistic insight into the transcriptional regulatory landscape of your system.

How Projects Run

All qPRO-seq projects are scoped individually to ensure the analysis is appropriate for the experimental design, biological question, and downstream goals.

​

  • Initial project planning meeting

An online discussion to review experimental design, spike-in strategy, sequencing depth, and analytical objectives before any processing begins.

  • Transparent, reproducible analysis

All analyses are performed using documented, reproducible workflows, with clear communication around key decisions such as spike-in normalisation, pause site calling parameters, and transcription unit identification strategy.

  • Results review and next steps

A final meeting to walk through results, visualisations, and biological interpretation, with guidance on follow-up analyses, validation strategies, or publication preparation.

Raw Data Processing

Starting from £45 per sample

  • Processing of paired-end qPRO-seq data using reproducible pipelines built with Conda, Python, and Snakemake

  • Alignment to the appropriate reference genome—and spike-in genome where applicable—using Bowtie2

  • Comprehensive quality control including FASTQC summaries, alignment metrics, strand specificity assessment, and run-on efficiency estimation

  • Filtering for properly mapped reads and removal of low-quality alignments

  • Spike-in-based normalisation for quantitative comparison across conditions

  • Generation of strand-specific, normalised BigWig files for interactive genome browser visualisation

  • Fixed software versions and fully documented parameters to ensure full reproducibility

  • Standard pipelines that can be customised to experimental design or institutional requirements

Basic Differential Gene Expression Analysis

Processing + basic analysis from £80 per sample

Includes everything in Raw data processing, plus:

  • Identification of transcription units (TUs) and bidirectional transcription sites using Tfit or FStitch

  • Quantification of nascent transcription at annotated gene bodies and promoter-proximal regions

  • Calculation of RNA Pol II pausing indices across conditions

  • Enhancer RNA (eRNA) identification at putative regulatory elements

  • Signal heatmaps and average profiles at transcription start sites and pause sites

  • Genome browser snapshots for representative loci

  • Clear, annotated results tables summarising transcriptional output by gene and region

  • Results meeting to discuss findings, interpretation, and biological relevance

Advanced Analysis

Processing + basic + advanced analysis from £120 per sample

Includes everything in Basic transcription analysis, plus:

  • Differential nascent transcription analysis between conditions

  • dREG-based identification of active regulatory elements from PRO-seq signal

  • Transcription factor motif enrichment at active regulatory elements

  • Analysis of transcriptional elongation dynamics and pause-release behaviour

  • Integration with ChIP-seq, ATAC-seq, or RNA-seq data where available

  • Pathway or gene-set enrichment analysis based on differentially transcribed genes

  • Iterative development of publication-ready figures and supplementary materials

  • Support with biological interpretation and results framing

 

​Packages are available for complete project support, from raw data processing through to publication-ready results.

All prices are indicative and may vary depending on experimental design, dataset size, species, and analytical scope. Academic and multi-sample project discounts are available.

bottom of page