STREAM 🌊

Streamlined Transcript Expression & RNA-seq Mapping

Nextflow DSL2 pipeline for RNA-seq quality control and transcript quantification. Ultra-minimalist — designed for solo bioinformaticians. Inspired by nf-core/rnaseq.

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Pipeline Overview

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flowchart TD
    subgraph INPUT ["Input (one of)"]
        SRA["SRR / ERR / DRR"] --> SRA_DL["SRA_DOWNLOAD"]
        GEO["GSE / GSM"] --> RESOLVE["RESOLVE_GEO"] --> SRA_DL
        FQ_DIR["FASTQ directory"]
        CSV["CSV samplesheet"]
    end

    SRA_DL --> FASTQS(("FASTQs"))
    FQ_DIR --> FASTQS
    CSV --> FASTQS

    FASTQS --> FQC1["FASTQC (raw)"]
    FASTQS --> FASTP["FASTP"]

    FASTP --> FQC2["FASTQC (clean)"]
    FASTP --> FQS["FASTQ_SCREEN (opt)"]
    FASTP --> SEQTK["SEQTK_STATS"]
    FASTP --> KRK["KRAKEN2 (opt)"]
    FASTP --> SALQ["SALMON_QUANT (opt)"]

    TX_DL["DOWNLOAD_TRANSCRIPTOME"] --> SIDX["SALMON_INDEX"]
    SIDX --> SALQ

    FQC1 --> MQC["MULTIQC"]
    FASTP --> MQC
    FQC2 --> MQC
    FQS --> MQC
    KRK --> MQC
    SALQ --> MQC

    MQC --> O1["MultiQC report"]

    classDef input fill:#0570b0,stroke:#0570b0,color:#fff
    classDef process fill:#238b45,stroke:#238b45,color:#fff
    classDef optional fill:#756bb1,stroke:#756bb1,color:#fff
    classDef output fill:#6a51a3,stroke:#6a51a3,color:#fff
    classDef data fill:#e6550d,stroke:#e6550d,color:#fff
    classDef mqc fill:#41ab5d,stroke:#41ab5d,color:#fff

    class SRA,GEO,FQ_DIR,CSV input
    class SRA_DL,RESOLVE,FASTP,FQC1,FQC2,SEQTK process
    class FQS,KRK,SALQ,TX_DL,SIDX optional
    class O1 output
    class FASTQS data
    class MQC mqc

Loading

Quick Start

# From a FASTQ directory (auto-detects PE/SE)
nextflow run IPNP-BIPN/STREAM --fastq_dir /path/to/fastqs --outdir results -resume

# From a samplesheet CSV
nextflow run IPNP-BIPN/STREAM --input samplesheet.csv --outdir results -resume

# From SRA accessions
nextflow run IPNP-BIPN/STREAM --sra_ids "SRR1234567,SRR1234568" --outdir results -resume

# From a GEO dataset (auto-resolves GSE → SRR)
nextflow run IPNP-BIPN/STREAM --sra_ids GSE123456 --outdir results -resume

# Full pipeline with all QC options
nextflow run IPNP-BIPN/STREAM \
    --fastq_dir /path/to/fastqs \
    --run_salmon \
    --run_fastq_screen --fastq_screen_conf /path/to/fastq_screen.conf \
    --run_kraken2 --kraken2_db /path/to/kraken2_db \
    --outdir results \
    -resume

Samplesheet format (CSV)

sample,fastq_1,fastq_2
sampleA,/path/to/sampleA_R1_001.fastq.gz,/path/to/sampleA_R2_001.fastq.gz
sampleB,/path/to/sampleB_R1_001.fastq.gz,

Leave fastq_2 empty for single-end reads. Multi-lane files with the same sample name are processed separately. To merge lanes, pre-concatenate or duplicate rows in the samplesheet.

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Parameters

Parameter	Default	Description
--input	null	Samplesheet CSV (sample,fastq_1,fastq_2)
--fastq_dir	null	Directory of FASTQs (*_R{1,2}_001.fastq.gz)
--sra_ids	null	SRA/GEO accessions (comma-separated or file, one per line)
--outdir	results	Output directory
--species	human	Species name (see supported species below)
--run_salmon	true	Enable Salmon quantification
--salmon_index	null	Pre-built Salmon index
--transcriptome_fasta	null	Transcriptome FASTA (skips download)
--genome	null	Genome assembly (auto-set from --species)
--ensembl_release	115	Ensembl release version
--run_fastq_screen	false	Enable FastQ Screen
--fastq_screen_conf	null	FastQ Screen config file
--run_kraken2	false	Enable Kraken2
--kraken2_db	null	Kraken2 database path
--fastp_qualified_quality	20	Minimum Phred score (fastp)
--fastp_length_required	20	Minimum read length after trimming
--skip_fastqc	false	Disable FastQC
--save_trimmed	false	Publish trimmed FASTQs
--subset_size	0	FastQ Screen subset (0 = all)
--max_cpus	auto	Maximum number of CPUs

Supported Species

--species	Organism	Genome Assembly
human	Homo sapiens	GRCh38
mouse	Mus musculus	GRCm39
rat	Rattus norvegicus	mRatBN7.2
zebrafish	Danio rerio	GRCz11
drosophila	Drosophila melanogaster	BDGP6.46
c_elegans	Caenorhabditis elegans	WBcel235

The transcriptome FASTA is automatically downloaded from Ensembl based on the species. You can also provide your own with --transcriptome_fasta.

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Output Structure

results/
├── 00_sra_fastq/        # Downloaded FASTQs (if SRA input)
├── 01_fastqc_raw/       # Raw reads QC
├── 02_fastp/            # Trimming reports + FASTQs (if --save_trimmed)
├── 03_fastqc_clean/     # Post-trimming QC
├── 04_fastq_screen/     # Contamination screening (optional)
├── 05_statistics/       # Sequence stats (seqtk)
├── 06_kraken2/          # Taxonomic classification (optional)
├── 07_salmon/           # Transcript quantification (optional)
├── 08_multiqc/          # Aggregated interactive report
├── reference/           # Transcriptome + Salmon index (cached)
└── pipeline_info/       # Nextflow timeline, trace, DAG, report

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Requirements

Core (always required): fastqc fastp multiqc seqtk

Optional: salmon (quantification) · fastq_screen bowtie2 (contamination) · kraken2 (taxonomy) · sra-tools pigz (SRA download)

Nextflow ≥ 23.04

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Resume & Cache

The pipeline natively leverages Nextflow's cache (-resume). Already completed steps are automatically skipped. References (transcriptome, Salmon index) are persisted via storeDir and reused across runs.

# Re-run after a crash — picks up exactly where it left off
nextflow run main.nf --fastq_dir fastqs --outdir results -resume

Mouse example

nextflow run IPNP-BIPN/STREAM \
    --fastq_dir /path/to/mouse_fastqs \
    --species mouse \
    --run_salmon \
    --outdir results_mouse \
    -resume

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License

MIT