BMC Genomics 2004, 5:65 http://www.biomedcentral.com/1471-2164/5/65 given the density of markers mapped and the distance between adjacent CS, new CS defined by the 7x sequenc- ing effort will be short in size and harbor a limited number of genes. What remains in canine genome map building is the development of a very high resolution gene map that can assist in the assembly of the 7x canine genome sequence now underway, and provide a mecha- nism for moving easily between canine and human com- parative segments. This is currently being undertaken. In the meantime, canine researchers can move forward with the continued development of the dog model for map- ping and cloning genes of interest to both human and companion animal health. Methods Markers and primer selection Canine BACs were randomly selected from the RPCI-81 canine BAC library [27] and were end-sequenced as described previously [1]. DNA was prepared using stand- ard automated approaches [31] and sequenced either at The Institute for Genomic Research (TIGR) or the Univer- sity of Washington High Throughput Sequencing Center. Average read lengths were in excess of 700 bp. Trace files representing BAC-end sequences were imported from ABI sequencers and examined for sequence homology to clon- ing vectors, Escherichia coli (E. coli) and repetitive DNA sequences. BAC sequences were also compared using Cross_Match with the complete E. coli genome to remove contaminating sequences of bacterial host origin. Finally, sequences were examined for interspersed repeats and regions of low sequence complexity using RepeatMasker http://ftp.genome.washington.edu/RM/RepeatMas ker.html. Primers defining each BAC-end were designed to regions of high quality sequence data using Primer3 software http://www-genome.wi.mit.edu/cgi-bin/primer/ primer3_www.cgi. Primers of 25 bp lengths were prefer- entially designed in order to minimize problems associ- ated with non-specific amplification, to generate amplicons of 200 to 500 bp, and to work under a single optimal set of PCR conditions. Genotyping Genotyping was performed using the RHDF5000-2 panel, which is comprised of 118 cell lines. The panel was con- structed by fusing dog fibroblasts irradiated at 5000 rads with TK-HTK3 hamster cells and has an experimental retention frequency of 22% with a theoretical resolution limit of 600 Kb [32]. PCR reactions were carried out at the University of Rennes and the Fred Hutchinson Cancer Research Center (FHCRC) as described previously in 15 µl volumes [1,33- 35] using the following touchdown program: 8 min 95°C, followed by 20 cycles of 30 sec 94°C, 30 sec 63°C decreasing by 0.5°C per cycle, 1 min 72°C and 15 cycles of 30 sec 94°C, 30 sec 53°C, 1 min 72°C and a final extension of 2 min 72°C. Primer pairs yielding either faint or spurious bands were rejected. PCR products were resolved and recorded as described [33,35] through a semi-automated data acquisition software. Quality control Duplicate genotypes were obtained for the 1092 new BACs added to the map. Data were considered consistent when the number of discrepancies between data sets was ≤ 16%, a value determined to correspond to a distance lower than the resolution limit of the RHDF5000-2 panel. In the rare cases where two independent typings yielded >16% discrepancies, a third typing was done and the resulting vector was either integrated into the map con- struction, or the marker was discarded if no agreement was observed between two of three genotypes. RH map construction RH vectors corresponding to the BAC-end markers and marker vectors of the previous map [1] were computed as a single data set using the MultiMap and TSP/Concorde algorithms [1,25,26,29]. Linkage groups were generated initially at a Lod score of 8.0, and where needed, at 9.0 to ensure strong statistical support. Individual linkage groups were analyzed using the multipoint approach of the rh_tsp_map version 2.0 of TSP/CONCORDE, as described previously [1,29]. Inter- marker distances are expressed in cR5000 units. Frame- work maps of well-spaced markers, supported by high quality data between adjacent markers, were initially gen- erated for all chromosomes. Distance criteria were set to 4cR, corresponding to the resolution capacity of the RH panel used, and quality criteria were fixed to 4, corre- sponding to a maximum allowable number of ambiguous data within RH vectors, as determined from previous analyses [1,35]. Distance and ordering of markers within each group was then determined by the TSP/Concorde and rh_tsp_map-2 algorithms [26,29]. Markers that could not be ordered with a high confidence level were submit- ted for re-analysis by stepwise increases in the Lod score to > 9.0, forcing the linkage group to split into two or more groups, until satisfactory order with a high statistical con- fidence level was achieved for each resulting group. Groups were then merged and oriented into a unique dataset. The merging step utilized the cytogenetic data obtained as part of this study, as well as the 2-point Lod scores between the markers at the extremes of each linkage group. Lod scores were generated using the pairlod_dist software from the rh_tsp_map package [26]. FISH mapping A total of 851 BAC clones from the RH map were also localized by multi-color FISH analysis. DNA from each Page 8 of 11 (page number not for citation purposes)