Lukacs et al. BMC Genomics 2010, 11:154 http://www.biomedcentral.com/1471-2164/11/154 fingerprint contig to represent core -and flanking BACs from each contig, followed by BAC DNA isolation described by CHORI. Positive BAC clone DNA from selected BACs were then PCR amplified with the SAA, UHA, ZE primers (Table 3) to verify that these BACs contained sequence of interest. 454 shotgun pyrosequencing The shotgun sequencing protocol using the 454 sequen- cing system (454 Life Science, USA) was performed according to manufactures protocol. Briefly, to generate the GS FLX shotgun library, the isolated Atlantic salmon BAC DNA was sheared into fragments, to which process specific A and B adaptors were blunt end ligated. After adaptor ligation, the fragments were denatured and clon- ally amplified via emulsion PCR, thereby generating mil- lions of copies of template per bead. The DNA beads were then distributed into picolitre-sized wells on a fibre- optic slide (PicoTiterPlate™), along with a mixture of smaller beads coated with the enzymes required for the pyrosequencing reaction. The four DNA nucleotides were then flushed sequentially over the plate. Light sig- nals released upon base incorporation were captured by a CCD camera, and the sequence of bases incorporated per well was stored as a read. DNA extractions were per- formed in our lab, while library generation and sequen- cing were performed at Royal Institute of Technology, Dept of Biotechnology (Stockholm, Sweden) and CEES, University of Oslo (Oslo, Norway). Assembly of the 454 reads were performed with Newbler. BAC shotgun library and sequencing The selected BACs were subjected to a shotgun sequen- cing approach and procedure is described in [13]. After sequencing run, bases were called using Phred [39,40]. High quality sequencing reads were assembled using Phrap [41], and viewed and edited using Consed [42]. Autofinish [43] was used for closing gaps by designing gap-closing primers with subsequent direct sequencing on BAC DNA or PCR amplification and PCR product sequencing. The BAC sequences were submitted to Genbank and given the following accession numbers: 114L13 [Genbank: FJ969488], 184H23 [Genbank: FJ969489], 439J08 [Genbank: FJ969490], 68O19 [Gen- bank: GQ505860], 439H13 [Genbank: GQ505859] and 129P21 [Genbank: GQ505858]. Bioinformatics DIGIT [44] and GENSCAN [45] were used to predict novel genes and to identify open reading frames. Dotter [46] was used to compare the BAC sequence to itself as well as to other BACs and to identify duplicated regions. Vista was used for sequence comparisons [47]. Blast searches identified possible functions of predicted genes Page 14 of 17 [48,49]. Sim4 [50] and Spidey [51] were used to adjust exon and intron boundaries aligning EST/cDNA sequences [26,27] to the BAC sequences. GRASP repeat- masker was used to identify repeats [52,53]. Multiple sequence alignments of the assumed or verified expressed exons were done using ClustalX [54] followed by manual inspection. TMHMM Server v. 2.0 was used for prediction of transmembrane domains for the MHC class I genes [55-57]. The amino acid sequences were aligned using Clustal W and inferred by ProTest [58] to find the best-fitting model of evolution (JTT, with an estimated alpha para- meter to 1.827, a gamma distribution of rates between sites of 4.0 and a proportion of Invariable Sites of 0.012). Phylogenetic analysis was performed using PhyML 3.0 [59], a fast and accurate maximum likelihood heuristic method, starting from the BIONJ tree under the para- meters estimated by ProtTest. Tree stability was assessed by means of a bootstrap analysis with 100 cycles. A phy- logenetic tree was also created using neighbour-joining method in MEGA version 4 [60]. Consensus trees were based on 1000 bootstrap replications and reported with the bootstrap support values (in percent) indicated above the respective nodes. Gaps were removed and phyloge- netic data reported using the Poisson correction model with uniform rates across all sites. Mapping the BACs 114I13 and 184H23 in Atlantic salmon The sequences of the BACs 114I13 and 184H23 were screened for suspected and known repetitive elements in salmonids using a salmonid-specific repeat masker [52]. The resulting sequences were subsequently searched for the presence of microsatellites using a Perl script created in Davidson Lab. 21 and 17 microsatellites were identi- fied in 114I13 and 184H23, respectively. Primer3 soft- ware [61] was used to design primers that would amplify the microsatellites containing the largest number of repeats. Several of these primer pairs were tested on the parents of the Atlantic salmon SALMAP mapping families, Br5 and Br6 [62,63], to determine which are informative. PCR amplifications were carried out in 6 uL reaction volumes in thin walled tubes using a Biometra T3 or T3000 Thermocycler. The reaction contained: 20 ng DNA, 1 × PCR buffer, 0.05 mM dNTP, 0.2 μM of the forward primer, 0.5 μM of the reverse primer, 0.5 μM of -21M13 primer (5’-TGTAAAACGACGGCCAGT-3’) labeled with HEX- or 6-FAM at the 5’ end, and 0.25 U of Taq polymerase (Qiagen). Touchdown PCR was per- formed as follows: a 2 min initial denaturation step at 94° C, then a cycle consisting of a denaturation step of 94°C for 30 seconds, annealing for 30 seconds and an elonga- tion step at 72°C for 30 seconds. The initial annealing temperature of 60°C was decreased by 0.5°C every cycle to 50°C, and then held at 50°C for an additional 14 cycles.