BACRB - Parallel Sequencing of Barcoded BAC Clones by random tagging PCR v1

A genome is assembled by connecting large scaffolds via a collection of thousands of mate-paired ends of various sizes (3-20 kbp). Terminal ends of long DNA fragments can be obtained by sequencing large insert clones by Sanger sequencing, a labour-intensive and expensive approach. Here we describe a high-throughput protocol to tag 11,520 BAC plasmids randomly with barcoded oligonucleotides at the end of a random hexamer by PCR.This method assisted the kiwifruit (Actinidia chinensis) genome assembly in a similar way as mate paired end datasets. Although not fully sequenced, each randomly tagged BAC clone provides a collection of short sequence tags mapped to a genome across one or multiple scaffolds under three different scenarios: validate a single scaffold, join two scaffolds, or group several scaffolds by proximity only.Our method provides a simple way of cataloging BAC libraries to assist genome assembly projects.