Fawcett et al. BMC Genomics 2011, 12:311 http://www.biomedcentral.com/1471-2164/12/311 in coding regions. We took advantage of the close evo- lutionary relationship between rhesus macaques and humans to generate hypotheses about the functionality of the non-synonymous SNPs we identified. With 411 SNPs that are predicted to be probably damaging by PolyPhen-2 [36], we have begun extending the informa- tion concerning potentially significant functional varia- tion in this species. Out of these SNPs, several occur in genes associated with behavior (Scn10A), metabolism (Ppgca1b), cancer (Brca2), and immune response (Map3k1, OAS2, IL18R1). Our study has identified substantial genetic variation among Indian-origin rhesus monkeys. Previous informa- tion [24] shows that separate SNP identification efforts are required in Chinese-origin and Indian-origin mon- keys. Only about one-third of SNPs that are poly- morphic in one population are also polymorphic in the other [27]. Thus, the information we report here is potentially helpful to investigators using Chinese-origin rhesus macaques, but additional work to discover SNPs in that population is warranted, just as information con- cerning variability among Chinese-origin animals is only partially relevant to our study population. While this study produced greater than 3,000 fold more SNPs than had previously been known in rhesus macaques (both Indian and Chinese), it is not exhaus- tive. Roughly 25% of each potential SNP list was vali- dated using our methodology (Figure S1, Additional file 1). Further validation of the remaining potential SNPs may proceed either through additional sequencing of the large data sets, or by sequencing additional animals. Currently, there are more than 37,821,067 human SNPs in dbSNP. Not only does that predict a lower bound of ~34 million rhesus macaque SNPs yet to be identified, but the hypothesized increased genetic variation in rhe- sus macaques indicates that we may identify many more SNPs than have been validated in humans. This study focused upon the genome-wide identification of SNPs in an effort to meet a wide variety of needs in the primate research community. Future work highlighting coding variants could take advantage of deep re-sequencing of exomes or re-sequencing specific loci where prior infor- mation suggests a potential role in disease or phenotype. Conclusions Our study identified more than 14 million potential sin- gle nucleotide variants and validated ~3 million of these variants. Various strategies can be used to detect and validate SNPs in any given species. We present an approach that combines different types of next-gen sequence data with low stringency bioinformatic filters, and relies upon differences among individuals and across sequencing chemistries to validate variants. Vali- dated SNP coverage was relatively even throughout the Page 9 of 13 genome. Additionally, a proportion of validated SNPs are predicted to be probably damaging in rhesus homo- logues of genes that are known to contribute to disease in humans. This increase in information concerning genetic variation in Indian-origin rhesus macaques opens up many new opportunities for the research com- munity in evolutionary studies, the use of rhesus maca- ques as models of human disease, and studies of primate diversity. Materials and methods Ethics Statement All relevant animal procedures in this project were approved by the appropriate Institutional Animal Care and Use committees from the Southwest National Pri- mate Research Center or the University of Wisconsin, and comply with the federal regulations. Genomic DNA for sequencing was obtained either from frozen tissues collected previously as part of other research projects or from blood samples collected from live animals by venu- puncture, using appropriate anesthetics. Data sources We began our analysis with the Sanger sequence data generated as part of the original rhesus macaque gen- ome project [23]. A single animal (animal ID 17573) from the Southwest National Primate Research Center (San Antonio, TX) Indian-origin rhesus colony was sequenced to approximately 5.2X coverage (18.4 Gb of raw sequence) using Sanger whole genome shotgun methods. Additional details of that reference genome analysis have been published previously [23]. From this Sanger data, approximately 4.3 million basepair posi- tions were called as potentially heterozygous using SNPdetector [40] (Houston, TX). We obtained addi- tional genomic DNA from animal 17573 and created two libraries for SOLiD sequencing, one library for frag- ment sequencing and one for mate-pair sequencing (Table S1, Additional file 3). Using published methods [41,42] (50bp fragment (SRA accession: SRX029055) and 25 × 25bp mate pair data (SRA accession: SRX029056 )) we generated 5X fragment sequence coverage and approximately 8X mate-pair sequence coverage. The proportion of matching beads to total beads was: 49.52% for the fragment library and 55.22% for the mate pair library (F3+R3). Following the re-sequencing of the ori- ginal reference animal, we selected two unrelated Indian-origin rhesus macaques (animal ID’s r1766 (SRA accession: SRX029057) and r02120 (SRA accession: SRX029058)) from the population at the Wisconsin National Primate Research Center (University of Wis- consin, Madison). Each DNA sample was used to pro- duce fragment sequencing libraries, and each library sequenced to low coverage (50bp HiDRA, r1766 to