RNAFramework Count Specifications

Process Details

• Name: RNAFramework_Count
• Process UUID: 3e7b31a7d72b4ef59e3c2a1a7160daa9
• Process Group: Misc.

Overview

The RNAFramework Count process implements the rf-count module of the RNA Framework, which enables RT-stop or mutations-per-position counting in transcripts. This process is essential for analyzing RNA structure probing data, particularly for SHAPE-MaP and DMS-MaPseq experiments, by quantifying chemical modifications and structural information at single-nucleotide resolution.

This process is implemented in Bash, which invokes the RNAFramework rf-count tool for mutation and coverage analysis.

Key Functionality

• Mutation Counting: Quantifies mutations per position in transcripts, supporting SHAPE-MaP/DMS-MaPseq analysis workflows
• RT-stop Analysis: Counts reverse transcription stops at each nucleotide position to identify RNA modifications
• Coverage Analysis: Calculates per-base coverage statistics without mutation counting
• Quality Filtering: Applies mapping quality, base quality, and read length filters to ensure data reliability
• Alignment Processing: Handles paired-end and single-end sequencing data with configurable alignment criteria

Input/Output Specification

Inputs

Required Inputs

• reads

  • Description: FASTQ file containing sequencing reads for analysis
  • Format: FASTQ

• genome

  • Description: Reference genome sequence in FASTA format
  • Format: FASTA

Optional Inputs

• bam_file
  • Description: Pre-aligned BAM files for direct processing
  • Format: BAM

Outputs

• directory

  • Description: Output directory containing all analysis results and intermediate files
  • Format: Directory

• mutation

  • Description: Mutation count files containing per-position mutation frequencies
  • Format: MM/RC

• rc_index

  • Description: Index file for rapid access to mutation count data
  • Format: Index

Parameters & Settings

These parameters can be adjusted in the Foundry UI when running this process.

• Primary alignments only

  • Description: Considers only primary alignments (SAM flag != 256)
  • Default value: false

• Paired alignments only

  • Description: When processing SAM/BAM files from paired-end experiments, only those reads for which both mates are mapped will be considered
  • Default value: false

• Properly paired alignments only

  • Description: When processing SAM/BAM files from paired-end experiments, only those reads mapped in a proper pair will be considered
  • Default value: false

• Mapping quality

  • Description: Minimum mapping quality to consider a read
  • Default value: 10

• Choose count method

  • Description: Coverage: Only calculates per-base coverage (disables RT-stops/mutations count); RT-stop: Standard RT-stop counting; Mutation: Enables mutations count instead of RT-stops count (for SHAPE-MaP/DMS-MaPseq)
  • Available options: Mutation (default), Coverage, RT-stop

• Raw Counts

  • Description: Generates a text file reporting raw (unfiltered) mutation counts, broken down by class (single nucleotide mutations, insertions, deletions)
  • Default value: false

• Specify mutations

  • Description: Only the specified mutations will be counted. Mutations must be provided in the form [original]>[mutated]. IUPAC codes are accepted. Multiple mutations must be provided as a comma or semi-colon separated list
  • Default value: G>Y;A>B;C>D;T>V

• Minimum length

  • Description: Discards reads shorter than this length (excluding clipped bases)
  • Default value: 1

• Minimum quality

  • Description: Minimum quality score value to consider a mutation (Phred+33)
  • Default value: 20

• Label low quality as not-covered

  • Description: If a mutated base does not exceed the minimum quality threshold, that base will be considered as non-covered
  • Default value: false

• Evaluate mutation surroundings

  • Description: When considering a mutation, also evaluate the quality of surrounding bases (± 1nt)
  • Default value: false

• No deletions

  • Description: Ignores deletions during analysis
  • Default value: false

• No insertions

  • Description: Ignores insertions during analysis
  • Default value: false

• Maximum deletion length

  • Description: Ignores deletions longer than this number of nucleotides
  • Default value: 10

• Maximum editing frequency

  • Description: Discards reads with editing distance frequency higher than this threshold (0<m<=1)
  • Default value: 0.15

References & Resources

• Tool Documentation: RNA Framework Documentation
• Related Papers: Incarnato, D., Neri, F., Anselmi, F., & Oliviero, S. (2014). RNA Framework: an all-in-one toolkit for the analysis of RNA-seq and structure probing data. Nucleic Acids Research, 42(21), e164. DOI: 10.1093/nar/gku959