High-Throughput Production of Randomly Tagged Barcoded BAC Insert End Pairs for Multiplex Illumina Sequencing

Genome assembly connects large scaffolds via mate paired ends encompassing an insert size between 3-20 kbp. Larger insert ends can be generated by sequencing BAC or fosmid clones ends by Sanger sequencing, a technique that in comparison with next generation sequencing methods is labour-intensive and expensive. Thousands of BAC and fosmid end sequence pairs are required to assist the assembly process. Here we describe a novel method for a high-throughput approach to randomly tag BAC plasmids with barcoded oligonucleotides at the end of a random hexamer by PCR, and a partition technique to retrieve only the paired BAC ends. Sequencing of BAC ends is then undertaken by NGS and bioinformatics scripts are used to deconvolute the data. We present a preliminary dataset using this method from 384 BAC clones derived from the kiwifruit (Actinidia chinensis) genome.