Opportunities and Challenges in Complex Genome Studies with Unbiased BAC Next-Gen Sequencing

The combination of large-insert bacterial artificial chromosome (BAC) and DNA sequencing technologies has been proved as the most powerful tool to dissect many complex genomes and or targeted genomic regions. However, traditionally BAC libraries built by restriction partial digestion are inherently biased with many genomic gaps. To completely cover interested-target genomic regions, finish genomes, or obtain the highest quality of genome sequencing assemblies of the extremely complex repeat-rich plant genomes, novel technologies are also needed to bridge the gaps between massively parallel short-read sequencing technologies.  We have successfully developed and continuously optimize novel technologies for unbiased large DNA isolation, purification, manipulation and Random Shear BAC cloning. We have also demonstrated that the Random Shear BAC libraries are unbiased and contains fewer gaps than conventional restriction enzyme based methods. Using this technology we are closing the gaps in the Arabidopsis genome.  We are able to sequence and assemble previously inaccessible regions of this genome by combining the new BAC library preparation methods with high depth short reads and long PacBio reads.  We will present the newest advances and accomplishments in BAC technologies and discuss the opportunities and challenges in complex genome assembly and analysis.