Phasing of single DNA molecules by massively parallel barcoding

• Published in: Nature communications Vol. 6; no. 1; p. 7173
• Main Authors: Borgström, Erik, Redin, David, Lundin, Sverker, Berglund, Emelie, Andersson, Anders F., Ahmadian, Afshin
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 09.06.2015; Nature Publishing Group; Nature Pub. Group
• Subjects: 38/47; 38/62; 631/208/325/2482; 631/337; 631/61/514/1948; DNA - chemistry; DNA Barcoding, Taxonomic; Humanities and Social Sciences; multidisciplinary; Science; Science (multidisciplinary)
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/ncomms8173

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, without losing phase information. DNA phasing information — the determination of which specific sequences belong to the same DNA molecule—is not easily obtained from sequencing applications that rely on short reads. Here the authors develop a phasing method based on massively parallel barcoding of single DNA molecules.