Abstract: Disclosed herein include methods of specifying sites (e.g., sites for colony formation) on a surface (e.g., a planar surface) and generating a flow cell having the sites specified on a surface. Also disclosed are methods of performing sequencing (e.g., sequencing-by-synthesis and sequencing-by-binding) using the flow cell generated and processing (e.g., aligning, orienting, sorting, and assessing quality) images of the flow cell captured during sequencing.