STARsolo

Overview

STARsolo is a module of the STAR aligner that provides a drop-in, open-source replacement for Cell Ranger’s gene expression quantification. It maps reads to a reference genome and demultiplexes cell barcodes and UMIs in a single pass, producing cell-by-gene count matrices compatible with downstream tools such as Seurat and Scanpy. STARsolo reproduces Cell Ranger results while offering substantially faster runtimes and greater flexibility for non-10x protocols.

Installation

mamba install -c bioconda star

Basic Usage

Align reads from a 10x Chromium 3’ v3 library and produce gene expression count matrices. Note that Read 2 (cDNA) is provided first, followed by Read 1 (barcode + UMI).

STAR --runMode alignReads \
  --genomeDir star_index/ \
  --readFilesIn sample_R2.fastq.gz sample_R1.fastq.gz \
  --readFilesCommand zcat \
  --soloType CB_UMI_Simple \
  --soloCBstart 1 --soloCBlen 16 \
  --soloUMIstart 17 --soloUMIlen 12 \
  --soloCBwhitelist 3M-february-2018.txt \
  --soloCellFilter EmptyDrops_CR \
  --soloFeatures Gene GeneFull \
  --outSAMtype BAM SortedByCoordinate \
  --runThreadN 16

Before running, build the STAR genome index if it does not yet exist:

STAR --runMode genomeGenerate \
  --genomeDir star_index/ \
  --genomeFastaFiles GRCh38.fa \
  --sjdbGTFfile genes.gtf \
  --runThreadN 16

Key Parameters

Flag / option	Description
`--genomeDir`	Path to the STAR genome index directory.
`--readFilesIn`	Input FASTQ files. For 10x data, provide the cDNA read first, then the barcode/UMI read.
`--readFilesCommand`	Command for decompressing input files (`zcat` for `.gz`).
`--soloType`	Barcode/UMI layout type (`CB_UMI_Simple` for standard 10x protocols).
`--soloCBstart` / `--soloCBlen`	Start position and length of the cell barcode within Read 1.
`--soloUMIstart` / `--soloUMIlen`	Start position and length of the UMI within Read 1.
`--soloCBwhitelist`	Path to the barcode whitelist (e.g., `3M-february-2018.txt` for Chromium v3).
`--soloCellFilter`	Cell-calling algorithm (`EmptyDrops_CR` matches Cell Ranger behaviour).
`--soloFeatures`	Which features to quantify (`Gene` for exonic, `GeneFull` for exonic + intronic).
`--outSAMtype`	Output format (`BAM SortedByCoordinate` produces a sorted BAM file).
`--runThreadN`	Number of threads to use for alignment.

Expected Output

STARsolo writes output into the default Solo.out/ directory:

Solo.out/Gene/filtered/ – filtered count matrix in MEX format (matrix.mtx, barcodes.tsv, features.tsv), directly loadable by Seurat or Scanpy.
Solo.out/Gene/raw/ – unfiltered matrix containing all detected barcodes.
Solo.out/GeneFull/filtered/ – count matrix including intronic reads (useful for single-nucleus RNA-seq).
Aligned.sortedByCoord.out.bam – coordinate-sorted BAM file with barcode and UMI tags.
Log.final.out – alignment summary statistics (mapping rate, unique vs. multi-mapped reads).