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Contributions to StringTie3

This fork contains development work for StringTie3's long-read module that was integrated into the main StringTie repository.

Publication

Shinder I, Pertea G, Hu R, Rudnick Z, Pertea M. StringTie3 Improves Total RNA-seq Assembly by Resolving Nascent and Mature Transcripts. Nature Methods (2025). [Accepted]

Preprint: bioRxiv 10.1101/2025.05.21.655404

Attribution Note

These contributions were developed in this fork (commits 23af2ba, e71e3c6) and integrated into the main repository by Geo Pertea in commit 29cd748, where he also fixed an edge-case bug he identified during testing. The commit message credits this work as "Ida's patch."

The full development history is preserved in this fork, and the implementation is described in the Methods section of the StringTie3 paper.

Commit History

Commits in This Fork (by Ida Shinder)

Commit Date Description Stats
23af2ba 2025-09-24 Feature/tts poly a graph (#3) - Main polyA implementation +1006, -157
e71e3c6 2025-09-26 Poly verbose v2 (#4) - Statistics and logging Additional refinements

Related Commit in Main Repository

Commit Author Description
29cd748 Geo Pertea "long read infinite loop fix for Ida's patch"

Lines of Code

Based on git blame analysis of this fork:

File Lines Contributed Description
rlink.cpp ~944 Core polyA detection, transcript boundary logic
rlink.h ~30 Data structure definitions
stringtie.cpp ~25 Global statistics variables
bundle.h ~14 Read alignment data structures
Total ~1,013

Long-Read Module Refinements

The long-read module refinements described in the StringTie3 paper (Methods: Long-read mode refinement) were developed in this fork.

Overview

1. Poly(A/T) Tail Detection

  • Scan up to 20 bp inward from alignment ends
  • Mark terminal aligned poly(A/T) when window contains ≥5 A/T bases and ≥80% A/T content
  • Handle both aligned segments and soft-clipped unaligned tails

2. Terminal Alignment Artifact Handling

  • Detect erroneous terminal exons from poly(A) tails splice-aligning to A/T-rich genomic loci
  • Discard single-exon alignments with terminal aligned poly(A/T) (frequent artifacts)
  • For multi-exon: shorten terminal exon to 3 bp to preserve splice junction

3. Poly(A) Site Anchoring (CPAS)

  • Cluster unaligned tail coordinates into 5 bp bins
  • Promote bins with ≥20 supporting reads to cleavage/poly(A) anchors
  • Constrain transcript boundaries during graph traversal

Functions Implemented

Function Location Purpose
poly_window_meets() rlink.cpp:510-545 Core A/T content checking
check_aligned_polyT_start() rlink.cpp:550-565 Aligned poly-T at read start
check_aligned_polyA_end() rlink.cpp:568-582 Aligned poly-A at read end
check_unaligned_polyA_end() rlink.cpp:585-600 Soft-clipped poly-A detection
check_unaligned_polyT_start() rlink.cpp:603-617 Soft-clipped poly-T detection
check_last_exon_polyA() rlink.cpp:619-637 Last exon A-fraction check
check_first_exon_polyT() rlink.cpp:639-656 First exon T-fraction check
shortenFirstExon() rlink.cpp:14118-14139 Trim first exon to 3 bp
shortenLastExon() rlink.cpp:14142-14163 Trim last exon to 3 bp
cluster_positions_with_counts() rlink.cpp:379-426 CPAS position clustering
add_cpas_trimpoint() rlink.cpp:428-447 Merge CPAS into graph anchors
ok_to_demote() rlink.cpp:362-370 Conservative junction demotion
junc_ratio_threshold() rlink.cpp:354-359 Tiered demotion thresholds
is_splice_between() rlink.cpp:450-453 Check splice gap between nodes
has_lr_witness_two_splices() rlink.cpp:483-506 Long-read validation for splice pairs

Constants Defined

POLY_TAIL_WIN         = 20    // Window size for aligned/unaligned end scan
POLY_TAIL_MIN_COUNT   = 5     // Minimum A/T bases in window
POLY_TAIL_MIN_FRAC    = 0.8   // Minimum A/T fraction (80%)
POLY_TAIL_MIN_CONSEC  = 5     // Minimum consecutive A/T bases
POLY_TAIL_STOP_COUNT  = 8     // Minimum reads for hardstart/hardend
CPAS_POS_BIN          = 5     // Cluster CPAS within 5 bp
CPAS_MIN_SUPPORT      = 20    // Require ≥20 reads per CPAS cluster

Data Structures Added

CTransfrag (rlink.h):

uint16_t poly_start_aligned;
uint16_t poly_end_aligned;
uint16_t poly_start_unaligned;
uint16_t poly_end_unaligned;

CReadAln (bundle.h):

uint16_t aligned_polyT;
uint16_t aligned_polyA;
uint16_t unaligned_polyT;
uint16_t unaligned_polyA;
int sort_tiebreaker;  // Stable sorting in mixed mode

CGraphnode (rlink.h):

bool hardstart:1;
bool hardend:1;
uint16_t polyStartAligned;
uint16_t polyStartUnaligned;
uint16_t polyEndAligned;
uint16_t polyEndUnaligned;

Global Statistics (stringtie.cpp):

g_longread_alignments_total
g_longread_singleton_screen_total
g_longread_singleton_discard_total
g_longread_removed
g_longread_unaligned_tail_total
g_longread_shortened

Development History

To explore the development history and implementation details, clone this fork:

git clone https://github.com/ishinder/stringtie.git
cd stringtie

# View commit history
git log --oneline --author="Ida"

# Count lines by author in key files
git blame rlink.cpp | grep -i "ida\|shinder" | wc -l

# View specific contributions
git blame -L354,430 rlink.cpp    # Constants and helper functions
git blame -L510,660 rlink.cpp    # PolyA detection functions
git blame -L14118,14165 rlink.cpp # shortenExon functions

# Show commit statistics
git show 23af2ba --stat

Syncing This Fork

This fork's master branch may be behind the main repository. The contributions described above have been integrated upstream. For the latest StringTie3:

git clone https://github.com/gpertea/stringtie
cd stringtie
make release

Contact

Ida Shinder