Finishing Working Draft BAC Projects by Directed Sequencing With ThermoFidelase and Fimers

A typical “working draft” bacterial artificial chromosome (BAC) project consists of approx 2000 shotgun reads obtained from a library of M13 or plasmid subclones that provide 2–5X coverage of BAC sequence. The reads are assembled into 5–50 long contigs (>2 kb), and hundreds of smaller contigs and singletons. The majority of the remaining gaps in the sequence are shorter than 1 kb. The utility of the working draft sequence is limited by the presence of the low-quality islands in the contigs, misassemblies, and by the presence of contaminating reads and contigs that originate from different BAC clones. One of the methods of finishing BAC projects consists of the production of additional shotgun libraries, obtaining 2000–4000 additional shotgun reads followed by the specialized finishing methods. The finishing methods require careful storage of the subclone libraries, optional shipping of them to the specialized facility, cherry picking of hundreds of subclones for end or directed sequencing, polymerase chain reaction (PCR) sequencing, and potentially even more specialized techniques.