STAR-suite

STAR-suite reorganizes STAR into module-focused directories while keeping a single source of truth for shared code. Build outputs remain compatible with existing STAR workflows, and the new top-level Makefile exposes module targets.

Folder Structure

core/
  legacy/                # Upstream STAR layout (single source of truth)
  features/              # Shared feature overlays (vbem, yremove, bamsort)
flex/                    # Flex-specific code + tools
slam/                    # SLAM-seq code + tools
build/                   # Modular make fragments
docs/                    # Suite-level docs
tests/                   # Suite-level tests
tools/                   # Suite-level scripts/utilities

Modules

• STAR-core (core/): Legacy STAR (indexing, bulk, Solo) plus shared utilities. Build: make core (binary at core/legacy/source/STAR).
• STAR-Flex (flex/): FlexFilter pipeline and Flex-specific integrations. Build tools: make flex or make flex-tools.
• STAR-SLAM (slam/): SLAM-seq quantification, SNP masking, trimming/QC. Build tools: make slam or make slam-tools.

Technical Updates

Core Updates

Recent updates to the Core module (STAR 2.7.11b and prior) include:

• Transcriptome Output: Replaced --quantTranscriptomeBan with --quantTranscriptomeSAMoutput for more explicit control (e.g., BanSingleEnd_ExtendSoftclip).
• Solo Features:
  • sF BAM tag for feature type and gene counts.
  • --soloCBtype String for arbitrary barcode strings.
  • Improved cell filtering and statistics with --soloCellReadStats Standard.

Flex Updates

STAR-Flex extends upstream STAR with features for 10x Flex, bulk RNA-seq, and reference management:

• Flex Pipeline: Inline hash-based processing for 10x Genomics Flex (Fixed RNA Profiling). Includes sample tag detection, 1MM pseudocount correction for CBs, clique-based UMI deduplication, and occupancy filtering.
• Cutadapt Parity: Native implementation of cutadapt v5.1 trimming algorithm (--trimCutadapt Yes) for bulk RNA-seq.
• TranscriptVB: Variational Bayes and EM quantification for transcript-level abundance, offering parity with Salmon alignment-mode.
• Reference Building:
  • AutoIndex: Automated reference download (--autoIndex), checksum verification (--autoCksumUpdate), and CellRanger-style formatting (--cellrangerStyleIndex).
  • Transcriptome FASTA: Automatic generation of transcriptome.fa during indexing for Salmon/TranscriptVB compatibility.
• BAM Sorting:
  • Samtools-style Sort: Spill-to-disk sorting (--outBAMsortMethod samtools) reduces memory usage compared to the default bin-based sorter. Configurable with --limitBAMsortRAM.
• Y-Chromosome Splitting: Split BAMs and FASTQs by Y-chromosome alignment (--emitNoYBAM, --emitYNoYFastq) for sex-specific analysis.

SLAM Updates

Integrated SLAM-seq quantification with GRAND-SLAM parity:

• Quantification: Full gene-level NTR estimation (Binomial/EM models).
• Compatibility Mode: --slamCompatMode gedi enables GEDI-compatible behaviors (intronic classification, lenient overlap, overlap weighting) for parity testing.
• Auto-Trimming: Variance-based detection of artifact-prone read ends (--autoTrim variance).
• QC: Comprehensive reports for T->C rates and error modeling.

QC Outputs

STAR-Flex and STAR-SLAM now generate detailed QC reports:

• SLAM QC (--slamQcReport <prefix>): Generates an interactive HTML report (.html) and JSON metrics (.json) visualizing:
  • T->C conversion rates per read position.
  • Variance analysis for auto-trimming (Stdev curves, segmented regression fits).
  • Trimming overlays showing chosen 5'/3' cut sites.
• FlexFilter QC (flexfilter_summary.tsv):
  • Cell calling statistics (EmptyDrops/OrdMag results).
  • Cell counts, UMI thresholds, and filtering rates per sample.

Summary of Flags

Core (Legacy)

Standard STAR flags apply. See core/legacy/README.md.

• --runMode: alignReads, genomeGenerate, soloCellFiltering
• --genomeDir: Path to genome index
• --readFilesIn: Input read files
• --outSAMtype: Output SAM/BAM format (e.g., BAM SortedByCoordinate)
• --soloType: Single-cell mode (e.g., CB_UMI_Simple, SmartSeq)

Flex

See flex/README_flex.md for full reference.

• Pipeline:
  • --flex yes: Enable Flex pipeline.
  • --soloFlexExpectedCellsPerTag: Expected cells per sample tag.
  • --soloSampleWhitelist: TSV mapping sample tags to labels.
• Trimming:
  • --trimCutadapt Yes: Enable cutadapt-style trimming.
  • --trimCutadaptCompat: Compatibility mode (e.g., Cutadapt3).
• Quantification:
  • --quantMode TranscriptVB: Enable VB/EM quantification.
• Y-Split:
  • --emitNoYBAM yes: Emit _Y.bam and _noY.bam.
  • --emitYNoYFastq yes: Emit split FASTQ files.
• Reference:
  • --autoIndex Yes: Enable automated reference download/build.
  • --cellrangerStyleIndex Yes: Use CellRanger-style reference formatting.
• Sorting:
  • --outBAMsortMethod samtools: Enable spill-to-disk sorting.

SLAM

See slam/docs/SLAM_COMPATIBILITY_MODE.md and slam/docs/SLAM_seq.md.

• Quantification:
  • --slamQuantMode 1: Enable SLAM quantification.
  • --slamGrandSlamOut 1: Generate GRAND-SLAM compatible output.
• Compatibility:
  • --slamCompatMode gedi: Enable GEDI compatibility.
  • --slamCompatIntronic, --slamCompatLenientOverlap: Fine-grained control.
• Trimming:
  • --autoTrim variance: Enable variance-based auto-trimming.
  • --slamTrim5p, --slamTrim3p: Manual trim guards.

Sample Commands

Core alignment:

core/legacy/source/STAR \
  --runMode alignReads \
  --genomeDir /path/to/genome_index \
  --readFilesIn reads.fq.gz \
  --readFilesCommand zcat \
  --outFileNamePrefix out/ \
  --outSAMtype BAM SortedByCoordinate \
  --outSAMattributes NH HI AS nM MD

Flex Mode (10x Fixed RNA Profiling):

core/legacy/source/STAR \
  --runMode alignReads \
  --genomeDir /path/to/flex_index \
  --readFilesIn reads_R2.fq.gz reads_R1.fq.gz \
  --flex yes \
  --soloType CB_UMI_Simple \
  --soloCBwhitelist /path/to/737K-fixed-rna-profiling.txt \
  --soloSampleWhitelist sample_whitelist.tsv \
  --outFileNamePrefix output/

SLAM Mode (Standard):

core/legacy/source/STAR \
  --runMode alignReads \
  --genomeDir /path/to/genome_index \
  --readFilesIn reads.fq.gz \
  --readFilesCommand zcat \
  --outFileNamePrefix out/ \
  --outSAMtype BAM SortedByCoordinate \
  --outSAMattributes NH HI AS nM MD \
  --slamQuantMode 1 \
  --slamSnpBed /path/to/snps.bed

SLAM Mode (GEDI Compatibility):

core/legacy/source/STAR \
  --runMode alignReads \
  --genomeDir /path/to/genome_index \
  --readFilesIn reads.fq.gz \
  --slamQuantMode 1 \
  --slamCompatMode gedi \
  --autoTrim variance \
  --outFileNamePrefix output/

More Detail

• Core usage: core/legacy/README.md
• Flex pipeline: flex/README_flex.md
• SLAM compatibility: slam/docs/SLAM_COMPATIBILITY_MODE.md
• SLAM methodology: slam/docs/SLAM_seq.md