Fawcett et al. BMC Genomics 2011, 12:311 http://www.biomedcentral.com/1471-2164/12/311 5.8X, r02120 to 3.3X). The ratio of matching beads to total beads was 58.64% and 49.89%, respectively. The library for r02120 exhibited a decreased degree of unique beads relative to r1766 and 17573, but did pro- duce a substantial amount of high-quality data. We also downloaded additional 454 sequence reads from NCBI (26.2 Mb) produced during the original rhesus sequencing project [23] from a comparison of rhesus maca- que sub-species (8 Chinese-origin and 8 Indian-origin unre- lated rhesus macaques). SNPs were called using these reads and the default settings for AtlasSNP2 [43] without filtering for coverage. Sub-species comparison read data was pro- vided at 0.5X coverage [23]. 142,781 potential SNPs were identified in the sub-species comparison data set. Several lists of SNP calls were generated previously (Table 3). MamuSNP provided a list of 22,892 potential SNPs [25,26](http://mamusnp.ucdavis.edu/query.php[44]). ENCODE consisted of a list of 1,672 validated SNPs out of which 1,467 have been previously described [24]. Addi- tional validated SNPs were downloaded from dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/[16]) and MonkeySNP (http://monkeysnp.ohsu.edu/snp/[45]), for a total of 765 SNPs at the time of download. Overlap was almost exact (763 of 765) between these two data sources and the data set will hereafter be referred to as dbSNP. SNP calling and validation methods Identification of specific bases that are heterozygous in a given individual was accomplished using different meth- ods for different sequencing technologies. The original Sanger read data for the reference animal was searched for SNPs (i.e. heterozygous basepair positions) using SNPdetector [40]. For the SOLiD read data, corona_lite v4.0 r2.0 (Life Technologies, Carlsbad, CA) was used. SNPs were called in the sub-species comparison data using AtlasSNP2 [43] as described above. Table 3 Data sources Chemistry Name Sanger Sanger SOLiD SOLiD SOLiD SOLiD 454 454 Assembly ENCODE 17573 fragment 17573 mate-pair r1766 r02120 Sub-species comparison MamuSNP Page 10 of 13 We considered all basepair positions called as heterozy- gous in any one dataset as “potential SNPs.” In order to classify a given SNP as “validated,” we required the specific base pair position be called as an identical heterozygote in two independent datasets. This could mean either obser- ving that position as heterozygous in the same individual using two different sequencing methods, or observing that position to be heterozygous, with identical alleles, in two different animals. Two complimentary methods were used to identify SNPs in the data sets described above: posi- tional-allele comparison and e-genotyping. Positional-allele comparison relies upon parameters of sequence quality and read coverage as implemented in var- ious SNP calling programs (in this study we used SNPde- tector, corona_lite, and AtlasSNP2). This method compares lists of identified potential SNPs by chromosome, base pair location, and both alleles. The comparison method can identify multiple SNPs clustered close together or those near to breakpoints in the reference sequence. But this method has a potentially higher false negative rate compared with e-genotyping, due to the relative stringency of the SNP calling programs used upstream. To reduce false negative calls, we used stringency thresholds some- what lower than those used in some other studies. Our set- tings generally retained potential SNPs with read quality scores equivalent to a Phred score of 20 and a minimum of 2 reads covering each variant allele call. E-genotyping is a novel approach that takes advantage of an a priori defined set of potential SNPs, and then tests raw reads (color-space or sequence space data) directly for exact probe matches to the previously defined SNP location and sequence. A probe region consists of 15 bases on each side of the potential SNP. These flanking regions must be pre-defined, and must match the target reads in the new sequence dataset exactly, with different reads providing exact matches to References Gibbs, et al. (2007) [23], Wheeler (pers. comm.) unknown dbSNP/MonkeySNP Unknown, >30 estimated Each data source is displayed with the appropriate chemistry used, DNA source (if known) and SNP calling software. Malhi, et al. (2007) [25], http://mamusnp.ucdavis.edu/query.php[44] http://monkeysnp.ohsu.edu/snp/[45], http://www.ncbi.nlm.nih.gov/ projects/SNP/[16] DNA source Animal 17573 ♀ Animal 17573 ♀ Animal 17573 ♀ Animal r1766 ♀ Animal r02120 ♂ ~7 pooled individuals SNP calling software SNPdetector Corona_lite Corona_lite Corona_lite Corona_lite Unknown Unknown 47 pooled individuals SNPdetector Hernandez, et al. (2007) [24] 16 pooled individuals AtlasSNP Gibbs, et al. (2007) [23]