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High-throughput sequencing platforms mainly produce short-read data, resulting in a loss of phasing information for many of the genetic variants analysed. For certain applications, it is vital to know which variant alleles are connected to each individual DNA molecule. Here we demonstrate a method for massively parallel barcoding and phasing of single DNA molecules. First, a primer library with millions of uniquely barcoded beads is generated. When compartmentalized with single DNA molecules, the beads can be used to amplify and tag any target sequences of interest, enabling coupling of the biological information from multiple loci. We apply the assay to bacterial 16S sequencing and up to 94% of the hypothesized phasing events are shown to originate from single molecules. The method enables use of widely available short-read-sequencing platforms to study long single molecules within a complex sample …