Parallel Sequencing of Barcoded BAC Clones to Assist and Validate Kiwifruit Genome Assemblies

A genome is assembled by connecting large scaffolds via a collection of mate-paired ends encompassing 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. Thousands of terminal end sequence pairs are required to assist the assembly process. We are sequencing a kiwifruit genome (Actinidia chinensis) using a combination of different NGS insert size libraries by Illumina and Roche 454 platforms. We also have a genomic DNA BAC library containing 69,120 clones. The library represents 7 times the genome (average BAC insert size is 71 kbp). We developed a high-throughput approach to tag 11,520 BAC plasmids randomly with barcoded oligonucleotides at the end of a random hexamer by PCR. Although not fully sequenced, each BAC clone provides a collection of short sequence tags mapped to a genome across one or multiple scaffolds under three different scenarios: validate a scaffold, join two scaffolds, or group several scaffolds by proximity only. We will discuss how this approach can assist the assembly of two kiwifruit genomes (Huang et al. 2013, and ours).