In CUTANATM Cloud, commonly used alignment outputs are conveniently located within the "Alignments" tab, designed for efficient access and streamlined downstream analysis. For each aligned reference genome, the platform generates a comprehensive suite of key files, empowering users to thoroughly evaluate experimental success and conduct in-depth investigations.
Key Alignment Output Files for Downstream Analysis:
Detailed Description of Bioinformatic Methods Used ("Alignments > Info > Run Methods"): Transparency is key. This section meticulously outlines the specific bioinformatic algorithms, parameters, and pipelines employed during the alignment process, ensuring reproducibility and facilitating a deeper understanding of the data generation.
Alignment Summary Report ("Alignments > QC Report"): This is an indispensable resource, serving as the central hub for evaluating key indicators of experimental success. The report provides a holistic overview, encompassing:
Seq Stats & Alignment Metrics: Detailed metrics on the alignment process, including mapping rates, duplicate reads, and other quality control indicators, are presented, often integrated with MultiQC for a consolidated view of multiple samples.
SNAP-CUTANA Spike-in results (if applicable): For experiments utilizing SNAP-CUTANA Spike-ins, this section provides crucial information on spike-in performance, which is vital for proper data normalization and interpretation. Further details on spike-in analysis can typically be found in the associated FAQs.
Heatmaps Illustrating Genome-Wide Target Enrichment: Visual representations in the form of heatmaps offer a quick and intuitive way to assess the enrichment of target regions across the genome, providing insights into the specificity and efficiency of the CUTANATM assay.
Interpretation of Results, Links to Files, and Next Steps: Beyond raw data, the report offers valuable interpretation of the alignment outcomes, guiding users through the implications of their results. It also provides direct links to relevant files for further exploration and suggests logical next steps for continuing the analysis workflow.
All Alignment Files ("Alignments > Files"): The robust alignment pipeline within CUTANATM Cloud generates a diverse array of files for each individual reaction, catering to various analytical needs:
Unique BAM Files: These represent the final, high-quality output of the alignment process. Unique BAM files contain reads that have been uniquely aligned to the reference genome and subjected to all necessary filtering steps, making them the primary input for downstream peak calling and differential analysis.
Unsmoothed BigWig Files (RPKM-normalized for heatmap generation): BigWig files are a compact and efficient format for displaying continuous data. The unsmoothed versions, normalized to Reads Per Kilobase per Million mapped reads (RPKM), are particularly useful for generating precise heatmaps that reflect genome-wide target enrichment without any artificial smoothing.
Smoothed BigWig Files (RPKM-normalized for visualization on IGV): For interactive visualization, smoothed BigWig files, also RPKM-normalized, are provided. The smoothing process enhances the visual clarity of enrichment patterns and makes them ideal for display and exploration using genome browsers like the Integrative Genomics Viewer (IGV), allowing researchers to easily identify enriched regions and compare profiles across samples.
TSS and Gene Body Heatmaps: Downloadable PNG files for the target enrichment heatmaps.
FASTQC Files: Although initial FASTQC analysis is often performed before alignment, the pipeline regenerates these analyses. This regeneration serves as a crucial reference for reviewing raw read quality control checks directly within the portal, ensuring data integrity from the outset.