Methods Oligonucleotide array CGH A custom 385k tiling-path array CGH was designed (Nimblegen Systems, http://www.nimblegen.com) to cover the preliminary Sus Scrofa assembly for chromosomes 4, 7, 14, and 17, from the August 2007 release (http://www.sanger.ac.uk/Projects/S_scrofa/), which was the newest release at the time of the experiment. The tiling-path array covers the chromosome sequences of the August 2007 release up to the old chromosome endpoint coordinates of the previous release (April 2007). The probe design fundamentals are described in the Nimblegen technical note http://www.nimblegen.com/products/lit/probe_ design_2007_11_13.pdf). Briefly, highly repeated elements in the genome were repeat-masked with a strategy similar to the Window- Masker program [40]. Concerning uniqueness, probes having a unique genome sequence match were selected with SSAHA [41]. An isothermal format (Tm = 76uC) [42] and probe length constraint between 50 and 75 bp were used for probe synthesis. The probes were integrated into an array design using ArrayScribeTM, which resulted in a design with a median probe spacing of 409 bp. The arrays were manufactured by maskless array synthesis technology and the oligonucleotides were synthe- sized on the arrays by photolithography [43–44]. Sample preparation From a pig family-material comprising 14 boar founders, 700 sows, and about 12.000 offspring, 12 of the Duroc boar founders (A, B, C, D, E, G, H, J, K, L, M, and N) were selected to function as test animals. An unrelated Hampshire boar was selected as the common reference. We adhered to our institutional guidelines for the ethical use and treatment of animals in experiments. Genomic DNA from boar N and the reference animal was isolated from lung/liver tissue by the use of Genomic-tip 100/G, Genomic DNA Buffer Set and Genomic DNA Handbook from Qiagen. After precipitation, the isolated DNA was resuspended in 16TE-buffer (10 mM Tris-HCl, 1 mM EDTA; pH 8). For the first 11 of the 12 test boars, genomic DNA was purified from blood to a concentration of ,250 ng/mL by salt precipita- tion. The precipitation was performed by adding 2.36volumes of cold 96% ethanol and 0.16volumes of 3M sodium acetate (pH 4.8). After 20 minutes of centrifugation at 4uC and 13.000 rpm, the precipitated DNA was washed with 70% ethanol, re-precipitated by 5 minutes of centrifugation at 4uC and 13.000 rpm. After precipitation, the isolated DNA was resus- pended in 16TE-buffer to a concentration of ,250 ng/mL. Following the isolation of the 13 genomic DNA samples, the DNA quality was assessed by measuring the concentration and the purity on the NanoDrop ND-1000 Spectrophotometer (Nano- Drop Technologies). DNA integrity and purity were also assessed by gel electrophoresis on a 1% agarose gel (SeaKemH GTGH Agarose, Cambrex Bio Science, and 16TBE buffer, Invitrogen) containing 0.15 mg/mL ethidium bromide. DNA fragmentation, labelling, and hybridization were carried out according to the manufacturer’s protocol. Briefly, the DNA was fragmented by sonication and labelled with Cy3- and Cy5- labelled 9mer primers. Each of the 12 boars was hybridized twice (technical replicates, 24 arrays) against the common reference with a MAUI hybridization system (BioMicro Systems). Scanning and intensity feature extraction were made as previous [45]. Statistical analysis The array data sets were imported into the R statistical programming language version 2.6.0 [46]. The intensity log2 ratios CNVs in the Pig Genome of the test versus reference samples were normalized with the Loess function from the Bioconductor [47] limma package [48]. In order to decrease the background noise of the arrays and retrieve only high-confident probes for each pair of technical replicate arrays, the probes that didn’t have a standard deviation of log2 ratio #0.2 were discarded while the others were averaged. The averaged normalized log2 ratios were used as input for the Bioconductor package snapCGH [49]. Segmentation of the data was then performed with three algorithms available in this package, DNAcopy [50], GLAD [51] and HomHMM [52]. The reason for using all these three algorithms instead of only one is that each one has a different approach to the segmentation scheme, providing both advantages and disadvantages in CNV detection [53–54]. Consequently, when using all these algorithms the false negative rate of CNVs detection is decreased, while the possible increase in false positives is addressed by using the following downstream filter criteria. CNVRs were called as the segments found by at least one of the mentioned algorithms with $5 consecutive probes, a median log2 ratio of 60.5 and detected in two or more animals. Subsequently, we used Tera-ProbeTM [55] an algorithm similar to the BLAST sequence search algorithm [56] but optimized for small oligonucleotide sequences. Tera-ProbeTM was used to query the probes within the CNVRs against the newest available version of the Sus Scrofa assembly (Sus Scrofa 6, May 2008 release), and probes were only kept if they had a unique optimal hit (100% sequence identity). Concerning this filtering criterion, we also kept the probes if there were two or more regions in the assembly that had a perfect hit from a block of at least half of the probes from a CNVR, since it was evident that these probes queried a putative segmental duplication. The CNVRs that decreased the number of probes to less than 5 were discarded. Finally, the remaining CNVRs were retained if they did not overlap any gap in the Sus scrofa 6, May 2008 release assembly. The NCBI’s pig Unigene database release 34 [57], based mainly on the data generated by the Sino-Danish Pig Genome Sequencing Project [58], the Refseq vertebrate mammalian mRNA database release 27 [57], and the Sus scrofa version 6, May 2008 release (http://www.sanger.ac.uk/Projects/S_scrofa/) was implemented to run on a DeCypher computer (http://www. timelogic.com). The Tera-BLASTTMN sequence similarity algorithm was used to query the CNVR sequences against the pig Unigene and the Refseq vertebrate mammalian mRNA databases. Hits were retained if they had an E-value #1e-15 and if their sequence aligned $95% (from Refseq) and $98% (from Unigene) with a CNVR. About 7k ,120 bp sequences around SNPs ascertained in- house (unpublished data) from the animals queried in the CNV study were also queried against the Sus scrofa version 6, May 2008 release with an E-value #1e-15 and they were retained if they had a perfect hit in the chromosomes 4, 7, 14 and 17. In order to check if the CNVRs overlapped any segmental duplication, Tera-BLASTTMN was used to query the CNVRs sequences, which are all above 1 kb in size, against the Sus Scrofa version 6, May 2008. Sequences were retained if they had . = 1 kb and . = 90% identity. The full data set from the oligo array CGH experiments has been submitted to GEO [59] under the accession ID GSE10753. Quantitative Real Time PCR Determination of copy number variation by quantitative real time PCR was performed using the Applied Biosystems 7900HT Sequence Detection System and analyzed with the SDS 2.2 PLoS ONE | www.plosone.org 7 December 2008 | Volume 3 | Issue 12 | e3916