Version Compatibility

Reference examples tested with: sra-tools 3.0+ (fasterq-dump, prefetch, vdb-validate, vdb-config), pysradb 2.2+, ENA portal API 2.0+

Before using code patterns, verify installed versions match. If versions differ:

• CLI: fasterq-dump --version, prefetch --version
• Python: pip show pysradb

If a flag is unrecognized or behavior changes, run <tool> --help and adapt.

SRA Data

"Download FASTQ from this SRA accession" -> Two paths exist in 2026: the SRA toolkit (NCBI's official, with prefetch + fasterq-dump) and the ENA mirror (EMBL-EBI's mirror with direct FASTQ download, often faster). For >1 TB workflows, a third path: AWS Open Data (STRIDES program) where same-region EC2 pulls SRA data with zero egress cost.

The single most impactful decision is where to pull from. SRA-direct is the default but ENA is faster more often than not, and AWS Open Data is the right answer for cloud-native analysis pipelines.

• CLI: prefetch SRR..., fasterq-dump SRR..., vdb-validate SRR... (sra-tools)
• CLI: curl https://ftp.sra.ebi.ac.uk/... (ENA mirror; direct FASTQ)
• CLI: aws s3 cp s3://sra-pub-run-odp/sra/SRR.../SRR... ./SRR....sra ... (STRIDES; object is unsuffixed; same-region free)
• Python: pysradb for metadata; subprocess for download

Required Setup

# sra-tools (toolkit)
conda install -c bioconda sra-tools           # 3.0+
fasterq-dump --version                        # confirm

# Configure cache location (default ~/ncbi/ -- often too small)
vdb-config --cfg                              # show current config
vdb-config --set /repository/user/main/public/root=/data/sra_cache

# Optional: pysradb for metadata
pip install pysradb

For STRIDES cloud:

# AWS CLI (no NCBI auth needed for public buckets)
aws s3 ls s3://sra-pub-run-odp/sra/SRR12345678/ --no-sign-request

Decision matrix: where to pull from

Default recommendation: ENA mirror for off-cloud, STRIDES (AWS/GCP) for in-cloud analysis. SRA-direct only when neither is available or when SRA format itself is needed (e.g. for re-extraction of technical reads).

SRA accession hierarchy

Conversion is via SRA metadata: pysradb metadata <ID> or efetch -db sra -id <UID> -rettype runinfo.

The actual download unit is SRR/ERR/DRR (runs). The BioProject (PRJNA...) is the convenient top-level handle for "pull all data for paper X".

fasterq-dump vs fastq-dump

fasterq-dump (sra-tools 2.10+) is the multi-threaded successor. Always prefer it, with two exceptions noted below.

The uncompressed-scratch trap: fasterq-dump writes uncompressed FASTQ first, then leaves it uncompressed. A 100 GB compressed FASTQ needs ~300 GB of scratch space + 300 GB of final output. Either compress post-hoc with pigz or use --mem to control RAM/disk tradeoff.

prefetch and the --max-size trap

prefetch downloads .sra files to the configured cache before extraction. Default --max-size 20G silently skips runs larger than 20 GB.

# Wrong: silently skips runs >20 GB
prefetch SRR12345678

# Right: set max-size explicitly to your largest expected size
prefetch SRR12345678 --max-size 100G -p

For unknown-size queues, set max-size to a generous upper bound (e.g. --max-size 200G) or query metadata first with pysradb metadata.

ENA mirror: direct FASTQ URLs

ENA stores FASTQ files directly (no SRA-format intermediate). Discover URLs via the ENA portal API:

curl 'https://www.ebi.ac.uk/ena/portal/api/filereport?accession=SRR12345678&result=read_run&fields=fastq_ftp,fastq_md5,read_count&format=tsv'

Returns TSV with semicolon-separated paired-end URLs and md5 checksums.

Direct download:

curl -O 'https://ftp.sra.ebi.ac.uk/vol1/fastq/SRR123/078/SRR12345678/SRR12345678_1.fastq.gz'

ENA's mirror is typically faster than SRA's because (a) it's hosted on Aspera-aware servers, (b) the FASTQ is pre-compressed (no SRA->FASTQ conversion needed), (c) EMBL-EBI's bandwidth is generous. For most downloads in 2026, ENA is the right default.

Single-cell / 10x quirks

10x Genomics records include "technical reads" (cell barcodes, UMIs) interleaved with biological reads. Default fasterq-dump (or fastq-dump) skips them. To get all reads:

# fasterq-dump with technical reads
fasterq-dump SRR12345678 --include-technical --split-files -p -O ./fastq/

# Some 10x records require fastq-dump -- check sra-stat first
sra-stat --xml SRR12345678 | grep -E '(spotCount|baseCount|tag)'

For 10x v3, expect 3 files per run: R1 (barcode+UMI), R2 (cDNA), I1 (index). For 10x v2: R1 (barcode), R2 (UMI+cDNA), I1.

MD5 / vdb-validate

Always verify downloads.

# vdb-validate for SRA-format files (toolkit path)
vdb-validate SRR12345678

# md5sum for ENA FASTQ files
md5sum -c <(echo "<expected_md5>  SRR12345678_1.fastq.gz")

ENA provides md5 in the portal API response. SRA-toolkit's vdb-validate is the equivalent for .sra files (different file format).

Cloud (STRIDES) access

NCBI's STRIDES initiative mirrored SRA data to AWS Open Data (us-east-1) and GCP (us-central1). Same-region pulls have zero egress cost.

# List SRA cloud-hosted files (no NCBI auth needed)
aws s3 ls s3://sra-pub-run-odp/sra/SRR12345678/ --no-sign-request

# Direct copy to EC2 in us-east-1. The STRIDES object is named without a `.sra`
# suffix (just SRR12345678); rename on copy to keep fasterq-dump happy.
aws s3 cp s3://sra-pub-run-odp/sra/SRR12345678/SRR12345678 ./SRR12345678.sra --no-sign-request

# Then fasterq-dump locally
fasterq-dump ./SRR12345678.sra -p -e 8

For cloud-native analysis pipelines (Nextflow on AWS Batch, Cromwell, etc.), STRIDES is the right path.

Code patterns

Single SRR via ENA mirror (preferred default)

Goal: Download paired-end FASTQ for one SRR; verify md5; minimal dependencies.

Approach: Query ENA portal API for FASTQ URLs and md5; download with curl; verify with md5sum.

Reference (ENA portal API 2.0+, curl):

#!/bin/bash
SRR="${1:-SRR12345678}"
OUT="${2:-./fastq}"
mkdir -p "${OUT}"

# Get FASTQ URLs + md5 from ENA portal API
META=$(curl -s "https://www.ebi.ac.uk/ena/portal/api/filereport?accession=${SRR}&result=read_run&fields=fastq_ftp,fastq_md5&format=tsv" | tail -1)
URLS=$(echo "${META}" | cut -f1 | tr ';' '\n')
MD5S=$(echo "${META}" | cut -f2 | tr ';' '\n')

i=0
while read url; do
    fname="${OUT}/$(basename ${url})"
    expected_md5=$(echo "${MD5S}" | sed -n "$((i+1))p")
    echo "Downloading ${fname}"
    curl -sL -o "${fname}" "https://${url}"
    actual_md5=$(md5sum "${fname}" | awk '{print $1}')
    if [ "${actual_md5}" != "${expected_md5}" ]; then
        echo "MD5 MISMATCH ${fname}: expected ${expected_md5}, got ${actual_md5}"
        exit 1
    fi
    echo "  md5 OK"
    i=$((i+1))
done <<< "${URLS}"

prefetch + fasterq-dump (SRA toolkit, classic)

#!/bin/bash
SRR="${1:-SRR12345678}"
OUT="${2:-./fastq}"
THREADS="${3:-8}"
mkdir -p "${OUT}"

# prefetch with explicit max-size (default 20G silently skips larger)
prefetch "${SRR}" --max-size 100G -p

# Validate SRA file
vdb-validate "${SRR}" || { echo "Validation FAILED"; exit 1; }

# Extract FASTQ (multi-threaded; uncompressed scratch ~3x final size)
fasterq-dump "${SRR}" -O "${OUT}" -e "${THREADS}" -p --split-files

# Compress post-hoc (fasterq-dump does NOT compress)
pigz -p "${THREADS}" "${OUT}/${SRR}"_*.fastq

# Cleanup SRA cache if you don't need it
# rm -rf ~/ncbi/sra/${SRR}.sra

Batch via pysradb metadata

Goal: Convert a list of GSE / BioProject / SRX IDs to SRR run accessions.

Approach: pysradb metadata returns a full hierarchy table; pull SRR column.

Reference (pysradb 2.2+):

from pysradb import SRAweb
import pandas as pd


def gse_to_srr(gse):
    db = SRAweb()
    df = db.gse_to_srp(gse)
    if df.empty:
        return []
    srp = df['study_accession'].iloc[0]
    runs = db.srp_to_srr(srp)
    return runs['run_accession'].tolist()


def bioproject_to_runs(prjna):
    db = SRAweb()
    return db.sra_metadata(prjna, detailed=True)


def batch_resolve(ids):
    db = SRAweb()
    rows = []
    for id in ids:
        try:
            meta = db.sra_metadata(id, detailed=True)
            rows.append(meta)
        except Exception as e:
            print(f'{id}: {e}')
    return pd.concat(rows, ignore_index=True) if rows else pd.DataFrame()


# Resolve a GSE to all its SRRs
srrs = gse_to_srr('GSE123456')
print(f'GSE123456 -> {len(srrs)} SRRs')

Cloud (STRIDES) via AWS

#!/bin/bash
# Run from EC2 in us-east-1 for zero egress
SRR="${1:-SRR12345678}"

# Check if available on AWS Open Data
aws s3 ls "s3://sra-pub-run-odp/sra/${SRR}/" --no-sign-request

# Download .sra (then extract locally)
aws s3 cp "s3://sra-pub-run-odp/sra/${SRR}/${SRR}" "./${SRR}.sra" --no-sign-request

fasterq-dump "./${SRR}.sra" -p -e 8 --split-files
pigz -p 8 "${SRR}"_*.fastq

10x single-cell with technical reads

#!/bin/bash
SRR="${1:-SRR_10x_run}"
OUT="${2:-./fastq_10x}"
mkdir -p "${OUT}"

# Get all reads including technical (barcode/UMI/index)
fasterq-dump "${SRR}" --include-technical --split-files -p -O "${OUT}" -e 8

# 10x v3 expects: R1 (28-bp barcode+UMI), R2 (cDNA), I1 (sample index)
ls -la "${OUT}/${SRR}"_*.fastq
pigz -p 8 "${OUT}/${SRR}"_*.fastq

Failure modes

prefetch --max-size silent skip

• Trigger: Default 20 GB limit; run is 50 GB.
• Mechanism: prefetch returns success but downloads nothing.
• Symptom: vdb-validate or fasterq-dump fails because no file exists.
• Fix: Always set --max-size explicitly to a generous upper bound (e.g. 200G).

fasterq-dump scratch space exhaustion

• Trigger: Run is 100 GB compressed; scratch dir has 200 GB free.
• Mechanism: fasterq-dump writes ~300 GB uncompressed, fills disk.
• Symptom: "out of disk space" mid-extraction.
• Fix: Use a scratch dir with 4-5x the compressed size; or use --mem to trade memory for disk; or stick with fastq-dump --gzip (slower but lower scratch).

10x technical reads missing

• Trigger: Default fasterq-dump on a 10x record.
• Mechanism: Technical reads (barcodes, UMIs) are skipped by default.
• Symptom: Only the cDNA file (R2) appears; CellRanger / STARsolo errors.
• Fix: Add --include-technical; verify with sra-stat --xml first.

SRA-direct slowness during US business hours

• Trigger: Downloading from NCBI 9 AM-5 PM ET weekdays.
• Mechanism: NCBI bandwidth contention; institutional users have priority.
• Symptom: kbps-level download speeds.
• Fix: Switch to ENA mirror or AWS STRIDES; run outside US business hours.

Aspera deprecation

• Trigger: Old script using ascp against [email protected].
• Mechanism: NCBI retired public Aspera in 2019; ENA followed ~2023; only institutional accounts retain support.
• Symptom: Connection refused or auth fails.
• Fix: Switch to HTTPS (slower but works); for fastest cloud transfer use STRIDES (AWS/GCP).

Cloud egress costs surprise

• Trigger: STRIDES pull from EC2 in us-west-2 against bucket in us-east-1.
• Mechanism: Cross-region egress is charged.
• Symptom: Unexpected AWS bill.
• Fix: Match compute region to bucket region (us-east-1 for AWS, us-central1 for GCP).

vdb-config not persisted across containers

• Trigger: Docker container without persisted ~/.ncbi/user-settings.mkfg.
• Mechanism: Cache config is per-user, per-home; container rebuild loses it.
• Symptom: Cache fills container's small layer; download fails.
• Fix: Mount a host volume at ~/.ncbi/ and persist user-settings.mkfg; or set --temp and -O explicitly in commands.

Common errors

References

• NCBI. SRA Toolkit documentation. https://github.com/ncbi/sra-tools/wiki
• NCBI. STRIDES program. https://datascience.nih.gov/strides
• Leinonen R, Sugawara H, Shumway M; International Nucleotide Sequence Database Collaboration. (2011) The sequence read archive. Nucleic Acids Res 39:D19-D21.
• Cochrane G, Karsch-Mizrachi I, Takagi T; International Nucleotide Sequence Database Collaboration. (2016) The International Nucleotide Sequence Database Collaboration. Nucleic Acids Res 44:D48-D50.
• Choudhary S. (2019) pysradb: A Python package to query next-generation sequencing metadata and data from NCBI Sequence Read Archive. F1000Research 8:532.

Related Skills

• entrez-search - Search the SRA db for accessions before downloading
• geo-data - GEO Series often link to SRA; gds -> sra ELink
• read-qc/quality-reports - QC the downloaded FASTQ
• read-qc/fastp-workflow - Adapter trim downloaded FASTQ
• ncbi-datasets-cli - Modern bulk path for genome data (NOT for SRA reads)