primer-panel 0.6.1

PCR primer panel pipeline: CDS target generation, Primer3Plus-like primer design, in-silico specificity check

Primer Panel

Primer Panel is a Python CLI for building human hg38 PCR primer panels from gene symbols. It separates target planning, Primer3 primer design, and genome-wide in-silico PCR specificity checks into clear pipeline stages.

What It Does

Stage	Purpose	Main output
1. Target generation	Select coding transcripts from Ensembl and build CDS target windows	target_summary.tsv, targets.bed, targets.fa
2. Primer design	Run Primer3 with Primer3Plus-like defaults	primers.tsv, primers.xlsx
3. Specificity check	Run UCSC isPcr and classify genome-wide hits	primers_specificity.tsv, primers_unique.tsv

The pipeline targets CDS regions, not full exons or UTRs. Stage 1 creates the template and SEQUENCE_TARGET; Stage 2 records Primer3 design metrics; Stage 3 is the only stage that reports genomic PCR product coordinates.

Installation

Primer Panel does not bundle primer3_core or UCSC isPcr binaries.

# Create environment with all tools
micromamba create -n primer_panel -c conda-forge -c bioconda \
  python=3.11 primer3 ispcr ucsc-fatotwobit \
  requests openpyxl pyfaidx -y
micromamba activate primer_panel

# Install primer-panel itself
pip install primer-panel

Or for development: pip install -e .

Verify

primer-panel --doctor

Tool summary

Tool	Stage	Install	Required?
primer3_core	2	micromamba install -c bioconda primer3	Required
isPcr	3	micromamba install -c bioconda ispcr	Required
faToTwoBit	3	micromamba install -c bioconda ucsc-fatotwobit	Optional (auto-.2bit)
bigBedToBed	dbSNP	micromamba install -c bioconda ucsc-bigbedtobed	Optional (.bb support)
pyfaidx	1	pip install pyfaidx	Required
openpyxl	any	pip install openpyxl	Optional (XLSX)

Docker / Apptainer

FROM mambaorg/micromamba:latest
RUN micromamba install -y -c conda-forge -c bioconda \
    python=3.11 primer3 ispcr ucsc-fatotwobit \
    requests openpyxl pyfaidx && \
    micromamba clean -afy
RUN pip install primer-panel

Quick Start

# Stage 1 only: target coordinates, no genome needed
primer-panel --genes HFE HJV TFR2 SLC40A1 HAMP FTH1 \
  --stage targets --output-dir outputs/targets

# Full pipeline (default): targets + primers + specificity
primer-panel --genes HFE HJV TFR2 SLC40A1 HAMP FTH1 \
  --target-size 2700-3300 --primer-flank 500 \
  --genome-fasta /path/to/hg38.fa --output-dir outputs/full

# Finalize: select best unique primer per target
primer-panel-finalize --input-dir outputs/full \
  --output-dir outputs/panel_final --genome-fasta /path/to/hg38.fa

Common Options

Option	Default	Description
--genes	(required)	Gene symbols to process.
--stage	all	targets, design, specificity, or all.
--target-size MIN-MAX	2700-3300	CDS target extension size.
--primer-flank N	300	Flanking bases for primer search.
--genome-fasta PATH	—	hg38 FASTA (required for Stage 2+).
--annotation-gtf PATH	—	Local Ensembl GTF for offline annotation.
--common-dbsnp-bed PATH	—	dbSNP file (.bed or .bb). See dbSNP.
--ispcr-db PATH	auto	Database for isPcr (auto-.2bit if faToTwoBit installed).
--ispcr-ooc PATH	auto	Overused-tile file.
--prepare-ispcr-db	off	Force (re)create .2bit.
--make-ispcr-ooc	off	Create .ooc file.
--doctor	off	Run dependency checks and exit.

isPcr Acceleration

isPcr over a full genome is slow. A .2bit binary database makes it fast. The pipeline auto-generates it on first run if faToTwoBit is installed (one-time, ~1-3 min). If faToTwoBit is missing, it falls back to FASTA with a one-time hint.

Also supported: .ooc overused-tile files (auto-discovered), and .nib databases (legacy). No flags needed — everything is automatic.

To force rebuild or pre-create: --prepare-ispcr-db (see primer-panel --help).

dbSNP Annotation

--common-dbsnp-bed annotates primers with common variants (MAF ≥ 1%) from dbSNP, flagging primers that overlap high-frequency SNPs — especially at the 3' end where mismatches impair extension. Accepts .bed and .bb (bigBed) files.

Method 1: BED via UCSC table dump (dbSNP 151, easy)

# Download and convert (~9 MB compressed)
wget https://hgdownload.soe.ucsc.edu/goldenPath/hg38/database/snp151Common.txt.gz
zcat snp151Common.txt.gz | cut -f2,3,4,5 > snp151Common_hg38.bed

primer-panel --genes HFE HJV TFR2 --genome-fasta /path/to/hg38.fa \
  --common-dbsnp-bed snp151Common_hg38.bed --output-dir outputs/run

Method 2: bigBed directly (dbSNP 155+, requires bigBedToBed)

# Install once
micromamba install -c bioconda ucsc-bigbedtobed

# Download and use directly — no conversion (~2 GB)
wget https://hgdownload.soe.ucsc.edu/gbdb/hg38/snp/dbSnp155Common.bb

primer-panel --genes HFE HJV TFR2 --genome-fasta /path/to/hg38.fa \
  --common-dbsnp-bed dbSnp155Common.bb --output-dir outputs/run

Available builds: dbSnp153Common.bb, dbSnp155Common.bb. Check https://hgdownload.soe.ucsc.edu/gbdb/hg38/snp/ for newer releases.

snp151's ~6M common variants are generally sufficient — common variant calls (MAF ≥ 1%) are stable between builds.

Output annotation

primers_specificity.tsv includes: common_snp_risk (none/medium/high), *_common_snp_count, *_3p_common_snp_count, common_snp_hits.

Key Outputs

File	Stage	Description
target_summary.tsv / .xlsx	1	Coordinates, transcript, CDS coverage, QC status.
targets.bed	1	Design-template BED6.
required_regions.bed	1	Raw CDS regions that must be covered.
targets.fa	1	Template sequences (N-placeholder unless --genome-fasta).
failed_targets.tsv	1	Genes that could not be processed (if any).
primers.tsv / .xlsx	2	Primer3 pairs and design metrics.
primers_specificity.tsv	3	Primers + isPcr hit classification + dbSNP annotation.
primers_unique.tsv	3	Subset: unique_pass only.
stage3_summary.txt	3	Per-target specificity summary.
run_summary.txt	all	QC summary for the full run.

primer-panel-finalize

Selects the best unique primer per target from Stage 3 outputs.

primer-panel-finalize \
  --input-dir outputs/run \
  --output-dir outputs/panel_final \
  --genome-fasta /path/to/hg38.fa

File	Description
recommended_primers.tsv / .xlsx	Best unique_pass primer per target.
failed_or_needs_review_targets.tsv	Targets without a unique primer.
panel_summary.txt	Human-readable summary.

Rescue (experimental)

Re-runs Primer3 for targets without a unique primer. Must be explicitly requested. Currently only FTH1_cds1_4 has an implemented strategy.

primer-panel-finalize --input-dir outputs/run --output-dir outputs/panel_final \
  --genome-fasta /path/to/hg38.fa --rescue-target FTH1_cds1_4

See primer-panel-finalize --help for --rescue-all and --experimental-split-rescue.

Specificity Status

Status	Meaning
unique_pass	One hit, matching expected chromosome and coordinates.
unique_off_target	One hit, but not at the expected location.
multi_hit	Multiple genome-wide products.
no_hit	No product detected.
pcr_error	isPcr failed for the batch.

Dependency Doctor

primer-panel --doctor

Reports primer3_core, isPcr, faToTwoBit, bigBedToBed, genome FASTA, and common dbSNP status. Does not require --genes.

Offline Annotation (Ensembl GTF)

By default, Stage 1 queries the Ensembl REST API. For offline use (or to pin a specific release), provide a local Ensembl GTF:

primer-panel --genes HFE HJV TFR2 --stage targets \
  --annotation-gtf /path/to/Homo_sapiens.GRCh38.110.gtf.gz \
  --output-dir outputs/offline

GTF provides annotation only — for the full pipeline, also pass --genome-fasta. Use GTF and FASTA from the same Ensembl release.

Transcript selection priority: MANE_Select → Ensembl_canonical → longest CDS among protein_coding transcripts.

Development

python -m pytest
python -m compileall -q primer_panel tests
python -m build --no-isolation --sdist --wheel --outdir /tmp/primer-panel-build

License

MIT. See LICENSE.