rectify-rna 2.1.0

Unified RNA 3' End Correction Framework for poly(A)-tailed RNA sequencing

RECTIFY: Unified RNA 3' End Correction Framework

RECTIFY (RNA 3' End Correction Tool Integrating False-priming and poly(A) ambiguity) is a unified framework for correcting 3' end mapping artifacts in poly(A)-tailed RNA sequencing data.

Overview

RECTIFY addresses two fundamental problems affecting RNA 3' end mapping:

1. A-tract Ambiguity (Universal): Genomic A-tracts near true 3' ends create positional uncertainty affecting ALL poly(A)-tailed RNA-seq technologies
2. Technology-Specific Artifacts:
   • AG mispriming: Internal priming on A/G-rich regions (oligo-dT methods)
   • Poly(A) tail alignment: Tail bases align to genomic A-tracts creating systematic shifts (when poly(A) is sequenced)

Key Features

• Modular correction strategies that apply based on sequencing technology
• Universal A-tract ambiguity detection for all poly(A)-tailed RNA-seq
• AG mispriming screening (from original RECTIFY, Roy & Chanfreau 2019)
• Poly(A) tail trimming and indel artifact correction (for direct RNA-seq: nanopore, Helicos, QuantSeq)
• NET-seq refinement (optional, technology-independent)
• Unified output format with confidence scores and QC flags

How It Works

RECTIFY corrects common 3' end mapping artifacts through a series of modular steps:

┌─────────────────────────────────────────────────────────────────────────────┐
│  EXAMPLE 1: Homopolymer Deletion Artifact (Nanopore)                        │
│  ═══════════════════════════════════════════════════                        │
│                                                                             │
│  True RNA:     5'...GCTAAGCTTAAAAAA-3' + AAAAAAAAAA (poly(A) tail)          │
│                              └────┘                                         │
│                         6A genomic tract                                    │
│                                                                             │
│  Genome:          ...GCTAAGCTTAAAAAA|GTCACC...     (| = true CPA site)      │
│                                                                             │
│  Nanopore read:   ...GCTAAGCTT--AAAA|GTCACC        (2bp deletion in A-tract)│
│                             ↑↑                                              │
│                    systematic homopolymer error                             │
│                                                                             │
│  Problem:   Aligner maps 3' end 2bp upstream of true position              │
│             (deletion consumes genomic bases that should be in transcript)  │
│                                                                             │
│  RECTIFY:   Detects A-tract deletion, adjusts position +2bp                 │
│  Result:    Correct 3' end position restored                                │
└─────────────────────────────────────────────────────────────────────────────┘

┌─────────────────────────────────────────────────────────────────────────────┐
│  EXAMPLE 2: Multiple Indels Near 3' End                                     │
│  ═══════════════════════════════════════                                    │
│                                                                             │
│  Genome:          ...TACGTTTTTTAAAAAA|GTCA...                               │
│                          └────┘└────┘                                       │
│                          T-tract A-tract                                    │
│                                                                             │
│  Nanopore read:   ...TACGT---TTAA-AAA|GTCA                                  │
│                        ↑↑↑     ↑                                            │
│                   3bp del  1bp del                                          │
│                                                                             │
│  RECTIFY logic:                                                             │
│    • T-tract deletion (3bp): TRUE artifact → correct +3bp                   │
│    • A-tract deletion (1bp): TRUE artifact → correct +1bp                   │
│    • Total correction: +4bp                                                 │
│                                                                             │
│  Note: Insertions do NOT shift reference coordinates (no correction needed) │
└─────────────────────────────────────────────────────────────────────────────┘

┌─────────────────────────────────────────────────────────────────────────────┐
│  EXAMPLE 3: A-tract Ambiguity Window                                        │
│  ═══════════════════════════════════                                        │
│                                                                             │
│  Genome:    ...CGTACAAAAAAAA|GTCACC...                                      │
│                   └───────┘                                                 │
│                   8bp A-tract                                               │
│                                                                             │
│  Problem:   Any position within the A-tract could be the true 3' end        │
│             (indistinguishable from poly(A) tail)                           │
│                                                                             │
│             ...CGTACAAAAAAAA|     ← could be here                           │
│             ...CGTACAAAAAAA|A     ← or here                                 │
│             ...CGTACAAAAAA|AA     ← or here                                 │
│             ...CGTACAAAAA|AAA     ← etc.                                    │
│                                                                             │
│  RECTIFY:   Reports ambiguity window [pos-7, pos] with range=8              │
│             Confidence score reflects uncertainty                           │
└─────────────────────────────────────────────────────────────────────────────┘

┌─────────────────────────────────────────────────────────────────────────────┐
│  EXAMPLE 4: NET-seq Refinement                                              │
│  ═════════════════════════════                                              │
│                                                                             │
│  Genome:    ...CGTACAAAAAAAA|GTCACC...                                      │
│                   └───────┘                                                 │
│               ambiguity window                                              │
│                                                                             │
│  NET-seq:           ▁▂▃█▇▅▂▁                                                │
│  signal:               ↑                                                    │
│                    peak at -3                                               │
│                                                                             │
│  RECTIFY:   Uses NET-seq Pol II occupancy to identify most likely           │
│             termination site within the ambiguity window                    │
│                                                                             │
│  Result:    Position refined to NET-seq peak, confidence = HIGH             │
└─────────────────────────────────────────────────────────────────────────────┘

Pipeline Flow:
══════════════

  ┌──────────┐    ┌─────────────┐    ┌─────────────┐    ┌─────────────┐
  │  Input   │    │  Module 1   │    │ Module 2A/B │    │  Module 3   │
  │   BAM    │───▶│   A-tract   │───▶│  Poly(A) &  │───▶│   NET-seq   │
  │          │    │  Ambiguity  │    │   Indels    │    │ Refinement  │
  └──────────┘    └─────────────┘    └─────────────┘    └─────────────┘
                        │                  │                  │
                        ▼                  ▼                  ▼
                  ┌───────────────────────────────────────────────┐
                  │              Corrected 3' Ends                │
                  │   (position, ambiguity range, confidence)     │
                  └───────────────────────────────────────────────┘

Installation

From source (development)

git clone https://github.com/k-roy/RECTIFY.git
cd RECTIFY
pip install -e .

From PyPI (future release)

pip install rectify

Quick Start

QuantSeq (oligo-dT short-read)

rectify correct quantseq.bam \
  --genome sacCer3.fa \
  --annotation genes.gtf \
  --polya-sequenced \
  --output corrected_3ends.tsv

Nanopore direct RNA-seq with NET-seq refinement

rectify correct nanopore.bam \
  --genome sacCer3.fa \
  --annotation genes.gtf \
  --polya-sequenced \
  --aligner minimap2 \
  --netseq-dir churchman_bigwigs/ \
  --output corrected_3ends.tsv

Output Format

RECTIFY produces a TSV file with corrected 3' end positions and QC metrics:

read_id  chrom  strand  raw_position  corrected_position  ambiguity_min  ambiguity_max  ambiguity_range  correction_type  confidence  qc_flags
read001  chrI   +       147588        147585              147583         147588         5                polya_trim       high        PASS
read002  chrI   +       147593        147591              147591         147593         2                ag_mispriming    medium      AG_RICH

Module Architecture

RECTIFY applies corrections modularly based on your data:

1. Module 1: A-tract Ambiguity (always applied)

   • Identifies genomic A-tracts near 3' ends
   • Calculates ambiguity windows

2. Module 2A: AG Mispriming (when oligo-dT priming used)

   • Screens for downstream AG-richness
   • Flags likely misprimed reads

3. Module 2B+2C: Poly(A) Corrections (when poly(A) IS sequenced)

   • Models and trims poly(A) tails
   • Detects and removes indel artifacts

4. Module 3: NET-seq Refinement (optional)

   • Resolves ambiguity using NET-seq data
   • Assigns confidence scores

Citation

If you use RECTIFY, please cite:

Original RECTIFY (AG mispriming correction):

Roy KR, Chanfreau GF. Robust mapping of polyadenylated and non-polyadenylated RNA 3' ends at nucleotide resolution by 3'-end sequencing. Methods. 2020 Apr 1;176:4-13. doi: 10.1016/j.ymeth.2019.05.016. PMID: 31128237

RECTIFY 2.0 (unified framework):

Manuscript in preparation

License

MIT License - See LICENSE for details

Contact

• Kevin R. Roy - [e-mail]
• GitHub: k-roy/RECTIFY

Acknowledgments

• Original RECTIFY development supported by Chanfreau Lab, UCLA
• NET-seq data from Churchman Lab, Harvard Medical School