Three types of internal transcription start sites (TSSs) identified by TL-seq. (A) Ribosome footprint density aligned relative to annotated start codons for Ribo-seq from glucose-starved yeast. Ribosomes accumulate at initiation AUGs but not internal AUGs (inset). (B) Ribosome footprint density for internal TL genes whose first AUG is in-frame with the annotated start codon. (C) Ribosome footprint density for internal TL genes whose first AUG is out-of-frame with the annotated start codon. (Inset) The first in-frame AUG for these same peaks. (D) Misannotated N termini. RNA-seq, Ribo-seq, and TL-seq support a TSS starting internal to the annotated AUG. (E) N-terminal peak. TL-seq called a TSS just inside of the annotated ORF. RNA-seq and Ribo-seq support such an internal TSS. (F) TL-seq identified a second internal TSS.
