A polynucleotide is barcoded using a method whereby an isolated, individual polynucleotide is immobilized on a solid phase and stretched, targets are labeled using target-specific hybridization probes, and an individual label of an unamplified probe at each of the labeled targets is optically detected. The order of the labels is determined to form a barcode representation of the polynucleotide wherein the targets and their relative positions are represented.