Bulk RNA-seq Gene Expression Analysis

ROSALIND's Bulk RNA-seq Gene Expression Analysis is a cloud-based discovery platform designed to empower scientists across oncology, immunology, regenerative medicine, drug discovery, and related fields to analyze and interpret gene expression data without requiring programming or bioinformatics expertise. By accepting both raw FASTQ files and processed counts data, ROSALIND enables researchers of every skill level to move from raw sequencing data to deep biological insights in a single, interactive environment.

RNA-seq is a fast-growing Next Generation Sequencing (NGS) assay used to evaluate gene expression, alternative splicing transcripts, and fusions. Scientists use it to compare healthy and disease states, identify differentially expressed genes and biological pathways, and discover therapeutic targets. ROSALIND connects experiment design, quality control, differential expression analysis, and pathway exploration in a real-time collaborative environment, delivering same-day results with every experiment.

Discovery Capabilities

• Analyze RNA-seq, NanoString, and gene count data
• Record experiment design and custom biological attributes
• Capture metadata using NCBI BioSample attributes
• Perform covariate and batch corrections
• Set up comparisons using biological attributes
• Create gene filters to adjust cut-off parameters
• Download publication-ready figures and plots
• Discover gene signatures and create gene lists
• Explore pathway, disease, and drug knowledge bases drawing from more than 20 leading sources
• Conduct real-time collaboration and results sharing
• Re-use existing samples for new experiments
• Import NCBI Short Read Archive (SRA) and Gene Expression Omnibus (GEO) public data automatically
• Find patterns across comparisons with meta-analyses
• Perform multi-omic analyses across experiment and assay types

Five-Step RNA-seq Workflow

1. Experiment Design: Create a new experiment through a guided experience that records biological objectives, sample attributes, and analysis parameters. ROSALIND fully supports NCBI BioProject and BioSample models for metadata assignment, and allows scientists to define custom attributes. Researchers can begin with raw FASTQ files or import processed normalized or raw counts from another pipeline, providing flexibility regardless of data source. The platform integrates a broad library of sample and library preparation kits, automatically calibrating each analysis for strandedness, strand direction, unique molecular identifiers (UMIs), and adapters.
2. RNA-seq Quality Control: ROSALIND's Quality Control Intelligence automatically evaluates key metrics including Q30 scores, alignment rates, ribosomal content, duplicate rates, sample correlation, gene coverage, genomic regions, and PCA/MDS for all samples. Non-aligning reads are evaluated for contamination when low alignment is detected, and alerts are generated when ribosomal content is unexpectedly high. Researchers can identify and eliminate outlier samples before proceeding to the discovery phase, without needing to be experts in sequencing quality control.
3. Unlocking Results: After reviewing quality control, researchers unlock the experiment using Analysis Units (AU), generally calculated at one AU per single-sample FASTQ file. Account balances and options for acquiring additional AU are accessible directly from the unlock screen.
4. Analysis and Discovery: ROSALIND provides a comprehensive dashboard for differential expression analysis that goes beyond a standard CSV file output. Default filters begin with a fold change of 1.5 (up and down) and a p-Adjust of 0.05, with further automated adjustments as needed. Researchers can create unlimited custom filters using fold change and p-value parameters. The platform features interactive visualizations including customizable heatmaps, volcano plots, MA plots, box plots, and bar plots, as well as deep interpretation of top pathways, gene ontology, diseases, and drug interactions. New comparisons and cross-experiment, multi-omic meta-analyses can be added at any time within minutes of setup.
5. Collaborating and Sharing Results: ROSALIND Spaces provides virtual data rooms where scientists and collaborators worldwide can interactively explore shared experiments in real time, similar to working with Google Docs. All changes are instantly available to authorized participants, with real-time activity feeds and historical reports. Spaces participants can add experiments, explore pathways, change cut-offs, add meta-analyses, and create new comparisons within the shared environment.

Comprehensive Analysis Pipeline

• Import from FASTQ or processed counts files
• Adapter trimming to remove sequences from high-throughput sequencing reads
• Optimized for nearly every NGS library kit to maximize the value of every short read
• Intelligent quality control and score assessment with automated contamination detection
• Genome alignment using an ultrafast universal RNA-seq aligner
• Quantification and normalization of sample reads
• Calculation of read distribution, fold changes, dispersion, and p-values
• Automated gene clustering in heatmaps
• Functional enrichment analysis of pathways, gene ontology, domain structure, and other ontologies
• Secure storage of experiment results and raw data files

ROSALIND is available to pharma, biotech, and academic researchers through Enterprise, Professional, and Academic subscriptions, as well as specialized licenses for service providers, contract research organizations (CROs), and academic core labs. The platform is cloud-based, requires no local installation, and supports real-time global collaboration, making it suitable for distributed research teams and multi-site organizations.