ruSTAR

A Rust reimplementation of STAR (Spliced Transcripts Alignment to a Reference), the widely-used RNA-seq aligner originally written in C++ by Alexander Dobin.

Overview

ruSTAR aims to be a faithful port of STAR, matching the original behavior as closely as possible. It uses the same genome index format, accepts the same --camelCase command-line parameters, and produces compatible SAM/BAM output.

Current status: End-to-end single-end and paired-end RNA-seq alignment with splice junction detection, two-pass mode, chimeric alignment detection, and multi-threaded parallel processing.

Quick Start

Build

cargo build --release

Generate genome index

target/release/ruSTAR --runMode genomeGenerate \
  --genomeDir /path/to/genome_index \
  --genomeFastaFiles /path/to/genome.fa

Align reads

target/release/ruSTAR \
  --genomeDir /path/to/genome_index \
  --readFilesIn reads.fq \
  --outSAMtype SAM \
  --outSAMstrandField intronMotif \
  --outFileNamePrefix /path/to/output_

Paired-end alignment

target/release/ruSTAR \
  --genomeDir /path/to/genome_index \
  --readFilesIn reads_1.fq reads_2.fq \
  --outSAMtype SAM \
  --outFileNamePrefix /path/to/output_

BAM output

target/release/ruSTAR \
  --genomeDir /path/to/genome_index \
  --readFilesIn reads.fq \
  --outSAMtype BAM Unsorted \
  --outFileNamePrefix /path/to/output_

Two-pass mode

target/release/ruSTAR \
  --genomeDir /path/to/genome_index \
  --readFilesIn reads.fq \
  --twopassMode Basic \
  --outFileNamePrefix /path/to/output_

Accuracy Comparison vs STAR

Single-End (10k yeast reads, 150bp)

Alignment Rates

Metric	ruSTAR	STAR
Unique mapped	83.1%	82.6%
Multi-mapped	5.4%	7.4%
Soft-clipped reads	26.6%	26.0%
Splice rate	2.1%	2.2%

Position and CIGAR Agreement

Mode	Position agree	CIGAR agree	Splice rate
Normal (default)	95.8%	97.8%	2.2%

SAM Tag Agreement (position-matching reads)

Tag	Agreement	Notes
NH (hit count)	98.3%	144 differ from seeding differences
HI (hit index)	100%
AS (alignment score)	98.7%	2 same-CIGAR diffs, rest from different CIGARs
NM (edit distance)	97.7%	0 unexplained: diffs = indel bases (NM vs nM semantics)
FLAG (primary)	99.8%	22 strand flips on multi-mapper ties
NH (with secondary)	96.2%	339 differ from different seeding

Junction Statistics (SE)

Metric	ruSTAR	STAR
Shared junctions	42	72 total
ruSTAR-only junctions	6	--
Motif agreement (shared)	100%	--

Paired-End (10k yeast read pairs, 150bp)

Alignment Rates

Metric	ruSTAR	STAR
Unique mapped	81.3%	78.6%
Multi-mapped	5.9%	5.3%
Total mapped	87.1%	100%
Unmapped pairs	12.9%	0%

Per-Mate Agreement

Metric	Value
Per-mate position agree	95.7%
Per-mate CIGAR agree	97.1%
Per-pair both mates correct	93.3%

Junction Statistics (PE)

Metric	ruSTAR	STAR
Shared junctions	72	90 total
ruSTAR-only junctions	6	--
Motif agreement (shared)	100%	--

Note: PE unmapped rate (12.9%) is because each mate must align independently before pairing. STAR uses joint DP stitching with mate rescue, which recovers pairs where only one mate has a clear alignment. Joint DP is planned for Phase 16.6.

Supported Features

• Single-end and paired-end alignment
• SAM and unsorted BAM output
• Multi-threaded parallel alignment (--runThreadN)
• GTF-based junction annotation with scoring bonus
• Two-pass mode for novel junction discovery (--twopassMode Basic)
• Chimeric alignment detection for single-end reads (--chimSegmentMin)
• Post-alignment read filtering (--outFilterType BySJout)
• Splice junction output (SJ.out.tab)
• Gzip-compressed FASTQ input (--readFilesCommand zcat)
• SAM optional tags: NH, HI, AS, NM, XS, jM, jI, MD
• SECONDARY flag (0x100) on multi-mapper alignments
• Configurable output limits (--outSAMmultNmax)
• Bidirectional seed search (L-to-R and R-to-L)
• Junction boundary optimization (jR scanning for optimal splice motifs)
• Deterministic output (identical SAM across runs)

Known Limitations

• PE unmapped rate higher than STAR (12.9% vs 0%) -- needs joint DP stitching for mate rescue
• No coordinate-sorted BAM output (use samtools sort post-alignment)
• No Log.final.out statistics file (MultiQC/RNA-SeQC)
• No paired-end chimeric detection
• No --quantMode GeneCounts
• No --outReadsUnmapped Fastx
• No --outStd SAM/BAM (stdout output)
• STAR uses nM (mismatches only); ruSTAR emits both NM (edit distance) and nM (mismatches only)

Building from Source

Requires Rust 2024 edition (rustc 1.85+).

cargo build --release    # Release build
cargo test               # Run tests
cargo clippy             # Lint
cargo fmt                # Format

Development

The majority of ruSTAR's code was written by Claude Code (Anthropic's AI coding assistant), with technical direction, architecture decisions, and validation by the project maintainer.

License

MIT (matching the original STAR license)