DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

2. Draft Sequencing Procedures for Chromosome 16 Sequencing

Mark O. Mundt, David C. Bruce, Leslie Chasteen, Judith Cohn, Lynne Goodwin, Kristina Kommander, Chris Munk, Robert Sutherland, Norman Doggett, and Larry Deaven

Bioscience Division and DOE Joint Genome Institute, Los Alamos National Laboratory, Los Alamos, NM 87545

mom@telomere.lanl.gov

As the amount of human sequence that is publicly available increases, efficient use of tools to monitor sequencing progress becomes a more important issue. Our current strategy for monitoring chromosome 16 draft data produced by the JGI includes immediate collection of information on 1) sequence marker content using ePCR, 2) BAC end sequence overlap with BLAST, 3) E. coli contamination level, 4) short subclone level, 5) Q20 quality analysis, and 6) confirmation of suspected overlaps based on our maps. These data are measured after the first two plates of forward and reverse sequencing, and decisions are then made for the continuation and desired level of sequence coverage

Order and orientation of contigs becomes the main concern as the draft depth is increased. We present Java tools to assist in both detecting assembly and tracking/handling errors as well as to help order contigs when possible. These tools take advantage of the paired end relationships that are available from our double-end plasmid sequencing approach. Finally, we demonstrate the importance of proper BAC end orientation in choosing clones to extend sequence as well as in feeding information back to a more accurate mapping process.