Design from genome, BED, and GTF files

BED mode treats every BED row as a required interval. GTF annotation is optional but useful for preserving nearby genes and regulatory features in the GenBank output.

BED example

smfprimer design bed \
  --genome docs/examples/genome.fa \
  --bed docs/examples/targets.bed \
  --gtf docs/examples/annotations.gtf \
  --search-window 40 \
  --product-size 50:100 \
  --min-tm 30 \
  --optimum-tm 50 \
  --max-tm 80 \
  --output genome_primers.tsv

BED and output coordinates are zero-based and half-open. GTF coordinates are one-based and closed and are converted internally.

The genome FASTA must be uncompressed and have consistent sequence-line width within each record. smfprimer builds an in-memory access index; it does not write an index beside the reference.

TSS example

TSS mode accepts a file containing one gene_id or gene_name per line:

smfprimer design tss \
  --genome docs/examples/genome.fa \
  --gtf docs/examples/annotations.gtf \
  --genes docs/examples/genes.txt \
  --tss-upstream 5 \
  --tss-downstream 5 \
  --tss-policy all \
  --search-window 40 \
  --product-size 50:100 \
  --min-tm 30 \
  --optimum-tm 50 \
  --max-tm 80 \
  --output tss_primers.tsv

Upstream and downstream follow transcriptional orientation. Use --tss-policy longest to choose the longest transcript per requested gene.