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RNA-Framework Module Pipeline Specification

Pipeline Details

  • Name: RNA-Framework Module
  • Pipeline UUID: b780db2315004c02b836cb5d0903183c
  • Version: 1.0.1
  • View Pipeline:

Overview

RNA-Framework Module pipeline is designed for processing, analyzing, and visualizing RNA chemical probing data (e.g., SHAPE, DMS, MaP approaches). It provides a comprehensive, modular suite of tools that handle the complete workflow from raw sequencing reads to secondary structure prediction, including trimming, aligning, computing mutation/RT-stop profiles, generating reactivities, performing normalization, and producing structure-ready outputs.

Key Use cases:

  • SHAPE-MaP Data Analysis: Processing SHAPE chemical probing experiments with mutational profiling to determine RNA secondary structure constraints.
  • DMS-MaPseq Experiments: Analyzing dimethyl sulfate probing data to identify accessible adenine and cytosine residues in RNA structures.
  • RNA Structure Prediction: Integrating chemical probing reactivities with computational folding algorithms to predict experimentally-informed secondary structures.

Features

  • Modular Design: Supports customizable workflows with plug-and-play modules (rf-index, rf-map, rf-count, rf-norm, rf-combine, rf-fold) that can be turned on/off as desired.
  • Multiple Aligner Support: Includes both Bowtie and Bowtie2 alignment options optimized for chemical probing read characteristics.
  • Chemical Probing Specificity: Tailored algorithms for handling mutation signatures from different probing chemistries (SHAPE, DMS, etc.).
  • Comprehensive Quality Control: Implements FastQC and MultiQC for sequencing quality assessment and summary reporting.
  • Flexible Normalization Methods: Supports multiple scoring and normalization approaches (Ding et al. 2014, Rouskin et al. 2014, Siegfried et al. 2014, Zubradt et al. 2016).
  • Structure Prediction Integration: Incorporates both ViennaRNA and RNAstructure folding engines with experimental constraint integration.
  • Replicate Handling: Automated combining of multiple experimental replicates with correlation analysis.

Input/Output Specification

Inputs

Required

Sequencing Reads

  • Description: FASTQ files containing raw sequencing reads from chemical probing experiments.
  • Format: .fastq.gz
  • Example File Path: /path/to/input/sample.fastq.gz

Reference Genome/Transcripts

  • Description: FASTA file containing RNA sequences to be used as mapping references.
  • Format: .fasta
  • Example File Path: /path/to/reference/transcripts.fasta

Optional Inputs

Pre-built Bowtie Index

  • Description: Pre-computed Bowtie/Bowtie2 index files for reference sequences.
  • Format: Bowtie index files (.ebwt or .bt2)
  • Example File Path: /path/to/index/reference_index

Outputs

Reported Outputs

  • MultiQC Report:
  • Description: Comprehensive quality control summary across all samples and processing steps
  • Format: .html
  • Example File Path: /output/multiqc/multiqc_report.html
  • Visualization App: Web browser

  • Location: MultiQC folder

  • Normalized Reactivity Profiles:

  • Description: XML files containing normalized per-nucleotide reactivity values
  • Format: .xml
  • Example File Path: /output/rf_norm/transcript_reactivities.xml
  • Visualization App: RNA structure visualization tools

  • Location: rf_norm folder

  • Predicted Secondary Structures:

  • Description: RNA secondary structure predictions in dot-bracket notation or connectivity table format
  • Format: .db/.ct
  • Example File Path: /output/rf_fold/structures.db
  • Visualization App: RNA structure viewers (e.g., VARNA, RNAstructure)
  • Location: rf_fold folder

Supporting Outputs

  • Aligned BAM Files:
  • Description: Mapped sequencing reads in binary alignment format
  • Format: .bam

  • Example File Path: /intermediate/rf_map/aligned_reads.bam

  • Mutation Count Files:

  • Description: Per-nucleotide mutation and coverage statistics
  • Format: .rc (RNA Framework count format)

  • Example File Path: /intermediate/rf_count/mutation_counts.rc

  • FastQC Reports:

  • Description: Individual sample quality control reports
  • Format: .html
  • Example File Path: /intermediate/fastqc/sample_fastqc.html

Associated Processes

References & Additional Documentation