Construction and validation of an equimolar barcode library. (A) Structure of the barcode linker. The linker contains a variable sequence part that consists of triplets of random (N) nucleotides that generate barcode diversity, flanked by fixed nucleotides that facilitate barcode retrieval in deep sequencing results. EGFP, enhanced green fluorescent protein. (B) Schematic representation of barcode library production and barcoding protocol. (C) Frequency distribution of barcodes in a 500-barcode library, determined by polymerase chain reaction and deep sequencing. (D) Correlation between the number of barcodes in samples submitted for sequencing and the number of barcodes retrieved in deep sequencing results.