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Assess specificity with Bowtie 1

Specificity assessment aligns concrete expansions of each ordered primer to a Bowtie 1 index and identifies inward-facing products within the configured amplicon-size range.

Build an index

bowtie-build GRCh38.fa GRCh38

The second argument is the index prefix passed to smfprimer.

Add specificity to design

smfprimer design fasta loci.fa \
  --product-size 250:500 \
  --bowtie-index GRCh38 \
  --specificity-mismatches 2 \
  --specificity-max-expansions 256 \
  --output primers.tsv

The tool invokes Bowtie 1 only when --bowtie-index is supplied. Use --bowtie-executable /path/to/bowtie when it is not on PATH.

Interpret the columns

  • specificity_forward_hits and specificity_reverse_hits count individual primer alignments.
  • specificity_off_target_amplicons counts compatible background products, which is more informative than individual hits alone.
  • specificity_off_target_loci records coordinates and mismatch counts.
  • specificity_status=no_off_targets means no compatible background product was formed under the configured mismatch and size rules.

Degenerate primers are expanded before alignment. A primer with many IUPAC positions can exceed --specificity-max-expansions; increase the cap carefully because alignment work grows with the number of expansions.

Important

Specificity is defined by the supplied index. Use an index representing the actual experimental background, including an appropriate transformed reference when assessing converted DNA.