Read Orientation Guessing (Strand Guesser)

By default, Rastair determines OT/OB orientation from SAM flags, assuming "standard" Illumina paired-end reads. However, some datasets have read orientation metadata that is incomplete, inconsistent, or incompatible with expected TAPS pairing conventions. This is especially common in tagmentation-based library preparations, which can produce non-directional reads.

With --guess-read-orientation, Rastair instead infers OT/OB from mismatch motifs observed in each read. The strand guesser provides an evidence-based fallback so methylation/variant strand assignments can still be made.

Inference Algorithm

For each aligned read, Rastair scans mismatch positions against the reference. At each mismatch, it inspects both 2 bp windows that include the mismatch base:

• current + next base
• previous + current base

Motifs are counted on the read sequence as represented by htslib/reference orientation:

• TG evidence supports OT
• CA evidence supports OB

Assignment rule:

• TG > CA => OT
• CA > TG => OB
• tie or no evidence => deterministic pseudo-random split per read

The tie-break is deterministic, so repeated runs produce stable OT/OB assignments for the same read.

Scope

This option currently affects the main pileup-based call workflow.