title: Mouse Lineage 2, passage 6
design: Three different mice were infected intranasally with S. pneumoniae strain BHN97x (19Fx), which was allowed to colonize for three days and initiate lineages M1-M3. This is a serotype 19F strain that colonizes to high density and causes acute otitis media, but typically does not cause invasive disease). After three days of colonization, bacteria were recovered from the nasal passages via retro-tracheal lavage, allowed to expand overnight for 16 h on TSA-blood agar plates to recover bacterial populations, and subsequently harvested for genomic DNA extraction and re-infection of the next passage in the respective lineages. This methodology was repeated for a total of ten generations per mouse lineage, involving 30 total infections. Whole genome sequencing of the bacterial populations (to >300 coverage, enabling detection of any mutation at ~1% or greater) was undertaken at each passage and mutation frequencies were subsequently determined.