Quality Assessment of Finished BAC Sequences

After the sequence of a bacterial artificial clone (BAC) clone is finished, it is important to assess the completeness and accuracy of the consensus sequence that was constructed from the underlying shotgun sequencing and finishing reads. Not all sequencing projects require that the entire BAC clone be finished to a specific high standard. For instance, in cases in which a BAC clone overlaps an already completely finished clone, the region of overlap need not be finished to the level that the nonoverlapping segment is finished. However, for this chapter, we refer to finished sequence to mean the complete BAC clone from end-to-end finished to high quality and meeting or exceeding the finished sequence standards set by the publicly funded Human Genome Project consortium. Although using some type of quality assessment technique is important at all stages of the sequencing/finishing process, and can be useful in determining a point at which to stop finishing a clone, our group at the Stanford Human Genome Center (SHGC), as well as most other sequencing groups, use quality assessment methods primarily for determining the quality of and performing quality control on finished sequence. Many of these techniques rely on the use of the Phred/Phrap/Consed system to help identify problem areas in the sequence of a BAC clone. In this chapter, we discuss the generally accepted standards of finished sequence and then describe the computational examination of the finished sequence, coupled with some laboratory techniques, for this quality assessment/quality control process.