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Conversion chemistry

smfprimer always accepts the ordinary, unconverted top-strand reference. The --converted-strand option identifies which reference strand undergoes the experimental chemistry.

Deaminase workflow

With --workflow deaminase, every cytosine on the converted strand is treated as a possible C/T position:

  • A forward ordered primer reports Y for C/T.
  • A reverse ordered primer reports the complementary R for A/G.

Conversion workflow

With --workflow conversion, non-target cytosines become expected thymine. Cytosines in the selected protected context remain ambiguous:

  • --context cpg protects CpG cytosines.
  • --context gpc protects GpC cytosines.
  • --context both protects both groups.

How Primer3 sees the sequence

Primer3 requires a concrete design sequence. smfprimer uses the expected converted member of each mixture (Y as T, and its ordered-primer complement R as A) for Primer3 selection and thermodynamic metrics. It then restores the conversion-aware IUPAC bases at Primer3's selected coordinates.

Consequently:

  • forward_order_sequence and reverse_order_sequence are the sequences to review for ordering.
  • Tm, GC, and Primer3 penalties describe the concrete converted allele.
  • forward_degeneracies and reverse_degeneracies show how many ambiguous positions each ordered mixture contains.

Do not interpret a low Primer3 penalty as a specificity guarantee. Specificity is assessed independently against the chosen Bowtie index.