PEARS

Pipeline for gene-fusion searching in Rna Single-cell sequences

PEARS is a Nextflow DSL2 pipeline that detects gene fusions at single-cell resolution from 10X scRNA-seq data. It combines three complementary fusion-calling approaches — FUSCIA, Flexiplex, and Arriba — and assigns cell barcodes to each detected fusion event, producing per-cell fusion calls.

Pipeline overview

1. Reference preparation — Downloads genome FASTA and GTF annotation (or uses pre-built references).
2. Fusion target generation — Builds search targets from a known fusions list using the reference annotation.
3. Alignment — Aligns reads with STARsolo (chimeric-aware) and produces a BAM and single-cell count matrix.
4. Fusion detection - Calls fusions using FUSCIA, Flexiplex, and Arriba in parallel.
5. Formatting — Consolidates results into per-cell fusion call CSVs per tool, and a combined_fusions.csv merging all three. For Visium HD data, spatial bin barcodes are also written to combined_fusions_spatial.csv.

Requirements

• Nextflow (>= 22.10)
• Conda (environments are built automatically from env/pears_env.yml)

Usage

After cloning the repository, use the path to the local directory:

Running locally:

nextflow run /path/to/pears \
  --fastq_r1 "/path/to/Reads_R1.fastq.gz" \
  --fastq_r2 "/path/to/Reads_R2.fastq.gz" \
  --known_fusions_list "known_fusions.csv" \
  --protocol "10x-3prime-v3" \
  --genome_version "GRCh38+GENCODE44" \
  -profile "local" \
  -resume

Running on SLURM cluster:

nextflow run /path/to/pears \
  --fastq_r1 "/path/to/Reads_R1.fastq.gz" \
  --fastq_r2 "/path/to/Reads_R2.fastq.gz" \
  --known_fusions_list "known_fusions.csv" \
  --protocol "10x-3prime-v3" \
  --genome_version "GRCh38+GENCODE44" \
  -profile "slurm" \
  -resume

Running on SLURM will be much faster due to parallelization of the fusion detection step. The -resume flag allows you to continue from the last successful step if the pipeline is interrupted.

Tip

Nextflow also supports running directly from GitHub without cloning first: nextflow run DavidsonGroup/pears [arguments]. This always fetches the latest version from the main branch.

Reusing references

Tip

After running the pipeline for the first time, you can extract the references and index from the references/ and STAR_index/ folders in the output directory. Note that the STAR index is built with a specific --sjdbOverhang parameter according to the read length of the input FASTQ. If you want to reuse the same index, make sure that the read lengths of the new FASTQ files are the same as the original ones. If they differ, you should rebuild the STAR index with the appropriate --sjdbOverhang (read length - 1) for optimal performance.

See Pre-built or reusing reference overrides for the relevant arguments.

Arguments

Basic

Argument	Default	Description
--fastq_r1	—	Glob pattern or path to Read 1 FASTQ files (gzipped).
--fastq_r2	—	Glob pattern or path to Read 2 FASTQ files (gzipped).
--known_fusions_list	—	CSV file of known/candidate fusions to search for (see Known fusions list format).
--protocol	—	10x Chromium chemistry preset (see Protocol presets). Sets the barcode whitelist and UMI length automatically.
--genome_version	GRCh38+GENCODE44	Genome build to download. Available versions: GRCh38+GENCODE40 through GRCh38+GENCODE49.
--out_dir	pears_output	Directory for all pipeline outputs.
-profile	—	Execution environment: local or slurm.

Protocol presets

--protocol sets the barcode whitelist and UMI length for the given 10x chemistry. These values can be individually overridden with --barcode_include_list and --umi_len (see Read structure overrides).

Preset	Chemistry	UMI length	Barcode whitelist
10x-3prime-v2	3' Gene Expression v2	10 bp	737K-august-2016
10x-3prime-v3	3' Gene Expression v3/v3.1	12 bp	3M-february-2018
10x-3prime-v4	3' Gene Expression v4	12 bp	3M-3pgex-may-2023
10x-5prime-v2	5' Gene Expression v1/v2	10 bp	737K-august-2016
10x-5prime-v3	5' Gene Expression v3	12 bp	3M-5pgex-jan-2023
10x-3prime-visiumHD	Visium HD spatial transcriptomics	9 bp	visium-hd

Read structure overrides

--barcode_include_list and --umi_len override the corresponding values set by --protocol. If --protocol is omitted entirely, both must be provided.

Argument	Default	Overrides
--barcode_include_list	set by --protocol	Barcode whitelist. Path to a custom whitelist file (can be gzipped).
--umi_len	set by --protocol	UMI length in bases.

Pre-built or reusing reference overrides

By default, the pipeline downloads the genome specified by --genome_version and builds the STAR index automatically. To skip this, provide all three arguments below — --genome_version is then ignored.

Argument	Default	Overrides
--ref_fasta	downloaded	Genome FASTA. Must be provided together with --ref_gtf and --star_genome_index.
--ref_gtf	downloaded	GTF annotation. Must be provided together with --ref_fasta and --star_genome_index.
--star_genome_index	built from download	STAR genome index directory. Must be provided together with --ref_fasta and --ref_gtf.

Fusion discovery

Argument	Default	Description
--discover_fusions	false	Search for novel fusions in addition to those in --known_fusions_list. Uses Arriba to discover candidates automatically. If no --known_fusions_list is provided, discovery is enabled automatically.
--min_arriba_support	20000	Minimum number of supporting reads required for a novel fusion discovered by Arriba to be included. Lower values find more candidates but increase runtime.
--arriba_exclusion_file	—	Path to a gzipped Arriba blacklist file (.tsv.gz) used to filter out likely false-positive fusions. Bundled blacklists for common genomes are available from the Arriba releases page. If not set, no blacklist filtering is applied.

Tool parameters

Argument	Default	Description
--flexiplex_searchlen	20	Length of fusion junction sequence to search for (2x actual overlap).
--flexiplex_demultiplex_options	auto-generated	Flexiplex demultiplexing options string. When not set, auto-generated as -b "?{barcode_len}" -u "?{umi_len}" -e 1 -f 0 where barcode length is read from the whitelist file and UMI length comes from --protocol or --umi_len. Setting this explicitly overrides the auto-generated value.
--fuscia_mapqual	30	Minimum mapping quality for FUSCIA read extraction.
--fuscia_up	1000	Upstream search distance (bp) when no gene annotation is available.
--fuscia_down	1000	Downstream search distance (bp) when no gene annotation is available.
--visium_bin_size	8	(Visium HD only) Bin size in microns for spatial barcode conversion. Must be 2, 8, or 16. Converts the two-part Visium HD barcode to standard SpaceRanger format (e.g. s_008um_00241_00258-1) in combined_fusions_spatial.csv.

Resource parameters

Argument	Default	Description
--cpus	16	Number of CPUs to allocate per process.
--memory	128 GB	Memory to allocate per process.
--time	48h	Wall-time limit per process (e.g. 24h, 2d12h).

Known fusions list format

The --known_fusions_list input is a CSV with the following required columns:

Column	Description
fusion genes	Fusion gene pair separated by -- (e.g. BCAS4--BCAS3).
chrom1	Chromosome of gene 1 (e.g. chr20).
base1	Breakpoint position of gene 1.
strand1	Strand of gene 1 (+ or -).
chrom2	Chromosome of gene 2 (e.g. chr17).
base2	Breakpoint position of gene 2.
strand2	Strand of gene 2 (+ or -).

Additional columns (e.g. classification) are ignored. This format is compatible with JAFFA output. Fusions involving mitochondrial genes (MT-) are automatically filtered out.

Example:

fusion genes,chrom1,base1,strand1,chrom2,base2,strand2,classification
BCAS4--BCAS3,chr20,50795173,+,chr17,61368327,+,HighConfidence
RPS6KB1--VMP1,chr17,59914703,+,chr17,59839768,+,HighConfidence
SLC25A24--NBPF6,chr1,108161182,-,chr1,108470597,+,HighConfidence

Output

Results are written to --out_dir (default pears_output/):

File/Directory	Description
combined_fusions.csv	All three tools merged into a single file, with UMI counts per tool and in total (see Combined fusions format).
combined_fusions_spatial.csv	(Visium HD only) combined_fusions.csv with cell barcodes converted to SpaceRanger spatial bin format (e.g. s_008um_00241_00258-1).
fuscia_fusion_calls.csv	Per-cell fusion calls from FUSCIA.
flexiplex_fusion_calls.csv	Per-cell fusion calls from Flexiplex.
arriba_fusion_calls.csv	Per-cell fusion calls from Arriba.
STARsolo/	BAM alignment, index, and single-cell count matrix.
fuscia_out/	Per-fusion FUSCIA discordant read files.
flexiplex_out/	Per-fusion Flexiplex barcode files.
arriba_out/	Arriba fusion table and per-fusion barcode files.
fusion_targets.csv	Generated fusion target coordinates and sequences.
nextflow_report.html	Nextflow execution report.
nextflow_trace.txt	Nextflow process trace log.

Per-tool fusion calls CSV format

The three per-tool CSVs (fuscia_fusion_calls.csv, flexiplex_fusion_calls.csv, arriba_fusion_calls.csv) share the same format:

Column	Description
cell_barcode	10x cell barcode (CB tag) identifying the single cell. The trailing -1 suffix is stripped.
molecular_barcode	Unique Molecular Identifier (UMI / UB tag) distinguishing distinct RNA molecules from PCR duplicates within the same cell.
fusion	Detected gene fusion, formatted as GENE1--GENE2 (names taken from the --known_fusions_list input).

Example:

cell_barcode,molecular_barcode,fusion
CCACAAAAGGTTCTTG,CAGGGATCAGTA,JPH1--NCOA2
CAGGGCTCACTTGGGC,TGATAGGAATCG,JPH1--NCOA2
GTGTGGCGTGGCCCAT,GGTAATCAGCAA,KIAA1429--RP11-586K2.1

Each row represents one unique observation of a fusion transcript in a specific cell. Rows are deduplicated — each (cell_barcode, molecular_barcode, fusion) triple appears at most once. Multiple rows with different cell barcodes for the same fusion indicate independent cells harbouring that fusion. Multiple rows with the same cell barcode but different UMIs for the same fusion indicate multiple distinct fusion transcript molecules captured in that cell, providing stronger evidence. Fusions detected by more than one tool in the same cell are higher confidence and can be identified by cross-referencing the three CSVs, or by using combined_fusions.csv directly.

Combined fusions CSV format

combined_fusions.csv merges calls from all three tools into a single file, aggregating UMI counts per (fusion, cell) pair:

Column	Description
fusion	Detected gene fusion, formatted as GENE1--GENE2.
cell_barcode	10x cell barcode identifying the single cell.
total_umis	Total unique UMIs supporting this fusion in this cell across all tools.
arriba_umis	UMIs from Arriba only.
flexiplex_umis	UMIs from Flexiplex only.
fuscia_umis	UMIs from FUSCIA only.

Example:

fusion,cell_barcode,total_umis,arriba_umis,flexiplex_umis,fuscia_umis
BCAS4--BCAS3,CCACAAAAGGTTCTTG,3,1,2,1
BCAS4--BCAS3,CAGGGCTCACTTGGGC,1,0,1,0

Cells with support from multiple tools (non-zero values in multiple *_umis columns) represent the highest-confidence fusion calls.

Credits

Adapted from FUSCIA (Steven Foltz, 2019) and Flexiplex (Davidson et al., 2022).