BMC Genomics 2006, 7:283 http://www.biomedcentral.com/1471-2164/7/283 markers from the genetic map, and about half of the anchored scaffolds contain two mapped markers allowing them to be orientated. The data presented here will increase the number of scaffolds that can be assigned and orientated. Nevertheless it will be necessary to use addi- tional information such as fingerprinting or BAC skim data and physical maps, such as FISH based techniques, which in addition to comparative mapping data will help to finalize the assembly and yield a reliable sequence. Methods Sequencing of ESTs A non-redundant "unigene" set of ESTs was selected by oligo-nucleotide fingerprinting and clustering of cDNAs from a brain library (Herwig et al., manuscript in prepara- tion). This non-redundant cDNA clone set contains 23040 bovine clones grouped by sequence assembly of ESTs into 14989 unique cDNA clusters and singletons. The cDNA clones of the "unigene" set were amplified in a 384-well microplate format by PCR consisting of an initial denaturing for 2 min at 95°C, denaturing for 45 sec at 94°C, annealing and elongation for 4 min at 65°C in 30 cycles. PCR primers were complementary to the insert- flanking vector sequences. The PCR mix contained 5 pmol forward primer (GGA TCT ATC AAC AGG AGT CCA AGC TCA GCT), 5 pmol reverse primer (TCA CCA TCA CGG ATC CTA TTT AGG TGA CAC), 0.1 mM dNTP's, 1.5 M Betain, 1× PCR buffer, 0.1 mM Cresol Red and 1 U per reaction Taq DNA polymerase. PCR buffer consisted of 0.5 M KCl, 1% Tween20, 15 mM MgCl2, 350 mM Tris- Base, 150 mM Tris/HCl pH 8.3. PCR fragments were sub- jected to sequence analysis using BigDye-terminator chemistry (Applied Biosystems) and a 3700 DNA sequencer (Applied Biosystems). Average sequence read length was 750 bp. The individual EST sequence data were submitted to GenBank and are publicly available under accession numbers CO871676–CO897060. Primer design Maximum sequence information for annotation was achieved by aligning the EST data with available public cattle transcript sequences contained in the TIGR bovine gene index. TIGR clusters and corresponding ESTs cattle sequences produced here were aligned and the resulting 14989 cluster sequences (consensus) used for the subse- quent construction of primers. Cluster sequences were aligned with bovine genomic sequences and only those showing clear splicing were used to define the precise exon-intron boundaries for the final primer selection (see below). The primer design was carried out using dedicated soft- ware now in the public domain [32]. The software uses the nearest-neighbour method [33] to predict the compli- mentarily of primers and secondary structures (dimers, hairpin etc.) and is able to process large number of sequences in batches, picking primers in designated regions. To minimize the amplification of hamster DNA contained within the RH panel cell lines, primer pairs were designed with one primer within exon, the other within the adjacent intron or non-coding sequence. The primer design was standardized to achieve a maxi- mum of uniformity in amplification conditions. Primer details are available to the public in the ArkDB database [27]. Screening of the Roslin RH panel 2473 marker loci were successfully typed on the 94 cell lines of a 3000-rad bovine/hamster RH panel as described by Williams et al. [7]. Vectors of 262 AFLP markers [26] were added to the dataset. Resulting vectors for the 3966 marker loci used (including 1231 previously mapped loci [7]) are available in the Additional File 4 for download. RH data analysis RH vectors were assigned to chromosomes by analysing 2- pt linkage with mapped loci [7] using RH mapper [34]. Multipoint maps were constructed using the default algo- rithm of the Carthagene software [35]. The initial multi- point map was improved by an iterative process of inspec- tion of marker loci and removal and alternative addition of badly linked or disrupting loci. This process resulted in the removal of 122 loci that could not be reliably fitted into the chromosome maps with the highest probability. The best maps generated by this process were compared to the ComRad RH-map [7] and the MARC 2004 linkage map [3] and regions showing discrepancies examined in detail to identify the presence of problem markers. Marker positions on the maps are available from the ArkDB data- base [27]. Mapping of marker associated sequences against the bovine sequence assembly ESTs sequences used to design the primers for mapped loci were aligned with the assembled 6× bovine sequence assembly (Btau_2.0) using BLAST [36] and SPIDEY [37]. To filter out incorrect alignments the BLAST e-value was set to a maximum of 1e-20 and minimum percent identity to 90%. In addition, the relative length of the BLAST hit (i.e. coverage, or length of the hit divided by the length of the query sequence) had to be at least 80%. Where ambig- uous alignments were observed higher stringency filters were applied (sequence similarity higher than 97.5% and coverage higher than 90%). Diagrammatic representation of chromosomal maps Visual representation of map alignments was achieved using cMap [38]. Page 12 of 15 (page number not for citation purposes)