Skill: Download SRA

Use When

• A user wants to download FASTQ files from NCBI SRA given one or more SRR, SRX, SRS, or SRP accessions.
• A user has an accession list file and wants to batch-download all runs.
• A user wants to expand a BioProject (PRJNA) or SRA study (SRP) to its full run list and download all runs.
• A user wants paired-end reads split into R1 and R2 FASTQ files.
• A user wants to control output directory layout, thread count, or compression.
• A user wants to verify download integrity (MD5 / Vdb-validate) after prefetch.

Inputs

• Required:
  • one of:
    • single SRR/SRX/SRS/SRP/PRJNA accession
    • path to a plain-text file of accessions (one per line)
• Optional:
  • --outdir DIR — output directory (default: sra_downloads)
  • --threads N — parallel threads for fasterq-dump (default: 6)
  • --split-3 — split paired-end into R1/R2 plus unpaired (default; recommended)
  • --split-files — split into separate files per read (use when --split-3 drops unpaired reads)
  • --original-sra — keep the .sra file after conversion (default: deleted)
  • --skip-prefetch — pipe directly through fasterq-dump without intermediate .sra (uses more temp disk)
  • --gzip — gzip-compress output FASTQ files (default: on)
  • --min-spots N — skip runs with fewer than N spots (default: 0, no filter)
  • --max-size SIZE — skip prefetch if expected size exceeds SIZE bytes
  • --temp-dir DIR — temp directory for fasterq-dump (default: system temp)
  • --no-verify — skip vdb-validate after prefetch

Workflow

1. Resolve the input to a list of SRR accessions:
   • If the input is a file, read it line by line (skip blank lines and # comments).
   • If the input is a SRP/PRJNA/SRX/SRS accession, expand to SRR list:

     esearch -db sra -query "<accession>" | efetch -format runinfo | \
       awk -F',' 'NR>1 && $1!="" {print $1}'
     

   • If the input is an SRR, use it directly.
2. For each SRR in the list, run scripts/run.sh:
   • prefetch --output-directory "$OUTDIR/sra_cache" "$SRR" to download the .sra file.
   • Optionally run vdb-validate "$OUTDIR/sra_cache/$SRR/$SRR.sra" to verify integrity.
   • fasterq-dump --split-3 --threads "$THREADS" --temp "$TMPDIR" --outdir "$OUTDIR/$SRR" "$OUTDIR/sra_cache/$SRR/$SRR.sra".
   • Gzip the resulting FASTQ files: pigz -p "$THREADS" or gzip.
   • Remove the .sra cache unless --original-sra was requested.
3. For batch mode (scripts/batch_run.sh), loop over the accession list sequentially or in parallel controlled by --jobs N (GNU parallel / xargs).
4. Print a per-run summary: SRR, spots downloaded, output files, size on disk, elapsed time.
5. Write manifest.tsv listing: SRR, sample name, R1 path, R2 path (if PE), size (bytes), md5sum.

Output Contract

• Per-run output under <outdir>/<SRR>/:
  • <SRR>_1.fastq.gz and <SRR>_2.fastq.gz for paired-end (split-3)
  • <SRR>.fastq.gz for single-end
  • <SRR>_unpaired.fastq.gz for unpaired reads from split-3 (may be absent or empty)
• manifest.tsv in <outdir>/ with columns: run, sample, r1, r2, spots, size_bytes, md5_r1, md5_r2
• Per-run log files under <outdir>/logs/<SRR>.log
• accessions_failed.txt listing SRRs that could not be downloaded

Limits

• SRA Toolkit (prefetch, fasterq-dump, vdb-validate) must be installed and on $PATH.
• fasterq-dump requires substantial temp disk space (typically 6–10× the compressed FASTQ size); set --temp-dir to a partition with sufficient space.
• dbGaP controlled-access data requires a valid ngc token file; pass it as NCBI_SRA_AUTH_TOKEN or via prefetch --ngc <token.ngc>.
• Very large SRA runs (>500 GB) may time out on slow connections; consider using the SRA on AWS (aws s3 cp s3://sra-pub-run-odp/sra/<SRR>/<SRR>) as an alternative.
• --split-3 is the recommended default for paired-end data; some older SRA entries do not store paired information and will produce single-end output regardless.
• Common failure cases:
  • insufficient temp disk causing fasterq-dump to abort mid-run, leaving partial FASTQ files
  • rate-limiting or network timeout during prefetch on large runs; retry with exponential backoff
  • accession does not exist or has been suppressed by submitter
  • mixing SRX/SRS into fasterq-dump directly without first resolving to SRR accessions