Fig. 3.

Identification of duplicates in ribosome-profiling reads. (A) Compositions of mRNA and RPF libraries prepared with fixed linkers or two different randomization strategies. Unique reads (blue) are sequences only present once in the library regardless of randomized sequences. Amplification duplicates (red) are reads that cannot be identified as unique based on randomization. The remaining sequences appear multiple times, but can be distinguished by randomization of the DNA linker (green), the RT primer (yellow) or a unique combination of both (orange). (B) Composition of two replicates of RPF libraries (as in A) with fixed linkers or dual randomization using a high input (samples as in A) or a 2–6× lower input of the same monosomal RNA for ribosome profiling. Library pairs (high:low) were randomly downsampled to an equal number of reads to accurately reflect differences in library composition.