Graphical Abstract.

TISCA reliably identifies translation initiation sites (TISs) and provides insight into the mechanism of translation initiation at near-cognate codons. TISCA efficiently reduces the background noise in GTI-seq analysis. The absence and nonrelease of eIF3-seq footprints are mostly eliminated as noise. Red and blue circles show the 5′ end of a GTI-seq peak and an eIF3 release point, respectively. TIS candidates are located at +11 to +13 nt from the GTI-seq peak and at −22 to −26 nt from the eIF3 release point (top). TISCA identifies a substantial number of near-cognate initiation codons, most of which are translated in a manner dependent on eIF2–Met-tRNAi (bottom).