BAC Sudoku Sequencing and Closing Repeat Rich BAC Clone

Bacterial artificial chromosome (BAC) libraries are important resource for positional cloning, whole genome sequencing, comparative mapping and functional genomics. There are thousands of BAC libraries that could be put to further use if an economical NGS strategy were available to decode their contents. Surprisingly, multiplex sequencing of BACs is little more advanced than individually barcoding them for sequence analysis on 454 Roche or Illumina platforms, which is prohibitively expensive on any scale. Although BACs are 1/50 the size of microbial genomes, it is still challenging to assemble them into one complete, contiguous sequence. We have developed a suite of tools and strategies that circumvent the deficiencies of the short read NGS platforms to completely assemble BACs and dramatically improve the multiplex sequencing of their contents. New library construction methods produce long span 8Kb reads on the Illumina or Ion Torrent platforms. In conjunction with fragment libraries we are able to assemble and close repeat rich BAC clones from maize and mouse DNA. A simple 3D pooling strategy was used to sequence multiple BAC clones as part of a project to discover microbial small molecule pathways. In silico analysis identified which contigs contained potential polyketide small molecule pathways, and deconvolution of the multiplex pooling strategy identified the individual clone containing the genes of interest. The incorporation of long span NGS reads and a 3D multiplex pooling strategy is a cost effective way to identify individual BACs of interest in a large library.