Stephen Fodor, Ronald Davis and colleagues have developed a cDNA barcoding method to estimate the efficiency of library preparation for RNA sequencing (RNA-seq) and to allow for more accurate quantification of transcript levels (Proc. Natl. Acad. Sci. USA 111, 1891–1896, 2014). The method uses a two-step process to generate cDNA libraries in which each molecule of any given cDNA sequence is labeled with a unique combination of barcodes. The authors use their labeled libraries to obtain absolute quantification of mRNA species after sequence capture enrichment for targeted RNA-seq, thus improving the ability to detect low–copy number transcripts while adjusting for error introduced during the amplification steps. Finally, the authors demonstrate that standard procedures for creating cDNA libraries for use in RNA-seq have extremely low efficiency. By spiking in a set of 960 barcoded synthetic RNAs, they were able to track the loss of input RNA through multiple steps of library creation. Overall, approximately 2–3 copies of a transcript remained in the completed library for every 1,000 copies in the input sample. These results demonstrate the need for improved methods of cDNA library preparation.