442 HUMAN MUTATION 28(5), 441^450, 2007 MATERIALS AND METHODS Supplementary Data Supplementary Tables S1, S2, S3, and S4, and Supplementary Figures S1, S2, S3, and S4 are available online at http:// www.interscience.wiley.com/jpages/1059-7794/suppmat and pre- sent data essential to this analysis. DNA Samples DNA samples were obtained from the Coriell Institute for Medical Research (Camden, NJ) or were isolated from peripheral blood donated on an anonymous basis by consenting adults according to a protocol approved by the Institutional Review Board (at the Hˆopital Sainte-Justine). A total of 40 unrelated samples representing five population groups were used to search for DNA variants that were subsequently confirmed by genotyping of the same samples using allele-specific oligonucleotide (ASO) hybridization. Sample groups were as follows: 1) sub-Saharan Africans including Biaka and M’Buti Pygmies (NA10469-71, Na10492) and African- Americans (NA17101, NA17108-10); 2) Americans of Amerindian descent from Brazil, Guyana, and Venezuela (NA17311-12, NA17314-20); 3) Europeans, represented by French-Canadians and Central Europeans (from Bulgaria, Germany, and Poland); 4) East and Southeast Asians from China and Indochina (Cambodia, Laos, and Vietnam); and 5) Southwestern Asians, represented by samples obtained from individuals of Jewish and Arab descent. A total of 40 additional samples from which genotypes were also determined were only compared in the calculation of Hardy- Weinberg equilibria (HWE) and fixation index (Fst) estimates. They included: 1) Africans South of the Sahara (NA17341-48); 2) Americans of Amerindian descent from the Andes (NA17303-10); 3) Hungarians (NA15199-202) and Russians (NA13838, NA13849, NA13852, NA13876); 4) Chinese (NA16654, NA16688-89, and NA17014-18); and 5) Indo-Pakistanis (NA17021-29). DNA samples of three great apes—common chimpanzees, gorillas, and orangutans—from Granby Zoo (Institut de la statistique du Qu´ebec) were included in ASO hybridization as well, to infer the ancestral allele at every polymorphic site. Polymorphisms and Genotyping Polymorphisms were detected by dHPLC (WAVE System of Transgenomic; Transition Technologies Inc., Toronto, Ontario, Canada) and sequencing as described [Sinnett et al., 2006]. Briefly, typically seven amplicons of 300 bp were designed (Primer 3 Software [Rozen and Skaletsky, 2000]) to cover the 2-kb segment upstream of the transcription start site. DNA fragments were amplified by standard PCR, using 10 ng of genomic template, and the products were analyzed by dHPLC at a minimum of two different temperatures. The purported hetero- duplexes were subsequently sequenced (Applied Biosystems 3730 xl DNA Analyzer; Applied Biosystems, Foster City, CA) to identify the underlying variants. Allelic states of substitution and small insertion/deletion polymorphisms in the human and in the great apes samples were determined by dynamic ASO hybridiza- tion as described [Bourgeois and Labuda, 2004], using the ASO probes listed in Supplementary Table S2. To avoid transcription errors, the genotypes were read and entered into our database twice by two independent individuals, and only concordant readings were accepted. Statistical Analyses Haplotypes for each 2-kb segment were derived from the corresponding genotypes using the PHASE program v. 2.0 Human Mutation DOI 10.1002/humu (www.stat.washington.edu/stephens/software.html) [Stephens et al., 2001]. Haplotype networks were drawn manually, assisted by Phylogenetic Network Analysis Software (www.fluxus engineering.com) [Bandelt et al., 1999]. We used ARLEQUIN software, v. 2.0 (http://lgb.unige.ch/arlequin) [Schneider et al., 2000], DnaSP package v. 4.0 (www.ub.es/dnasp) [Rozas et al., 2003], and H-test software (www.genetics.wustl.edu/jflab/ htest.html) [Fay and Wu, 2000], to compute population parameters, population variance Fst, Hardy-Weinberg equilibria, summary statistics, and to carry out neutrality tests. Tajima’s D test statistics [Tajima, 1989] considers the difference between YS and Yp, normalized by the expected standard deviation of this difference. The first estimator, YS 5 S= Pn 1 1=i, i where n corresponds to the number of sampled chromosomes [Watterson, 1975], is only influenced by the number of segregating sites, S. The second estimator, Yp, representing the mean number of pairwise differences between individual sequences [Tajima, 1989], corresponds to the product of nucleotide diversity and the sequence length L such that Yp 5 pL. The number of sites, S , 1 i.e., the number of sites with a derived allele observed only once (i 5 1), provides the estimator YF 5 S1 [Fu and Li, 1993]. YF is used in conjunction with either YS or Yp to calculate test statistics DFu&Li and F, respectively, as well as related statistics D and F [Fu and Li, 1993]. The estimator YH, by Fay and Wu, corresponds to the sum of twice the squared frequency of the derived alleles [Fay and Wu, 2000]. The corresponding H-statistics comparing Yp with YH are particularly sensitive to selection sweeps and the effect of genetic hitchhiking [Maynard Smith and Haigh, 1974], whereby a positively selected variant simultaneously drives up the population frequency of its neighboring tightly linked alleles. The test statistics Fs [Fu, 1997] examine the difference in the number of the observed haplotypes with those expected given Yp, thus confronting the latter with Yk. Tests by Ewens-Watterson and Chakraborty [Chakraborty, 1990; Ewens, 1972; Watterson, 1978] based on the infinite allele (haplotype) model can be considered to some extent complementary; they confront Yk, estimated from the number of haplotypes with Yhom, estimated from the haplotype homozygosity, hom 5 1–G. Evaluating the significance of the Ewens-Watterson test, Arlequin implements the protocol described by Watterson [1978] and that by Slatkin [1994]. All neutrality tests were carried out using data obtained with an initially ascertained sample of 80 chromosomes, except in the Fay and Wu test, where an extended sample of 80 genotyped individuals was used. The Hudson-Kreitman-Aguad´e (HKA) test [Hudson et al. 1987]—considering the number of segregating sites, S, as well as nucleotide diversity, p, as a measure of locus diversity—was executed as previously described [Jaruzelska et al., 1999]. The reported Fsts and the results of the HWE test were obtained using genotypes of the extended sample of 80 individuals. Correction for multiple testing (neutrality two-tailed and HWE tests) was carried out according to Storey [2002] using a false discovery rate of 10% (http://faculty.washington.edu/jstorey/qva- lue/index.html). Divergence, d, between human and chimpanzee sequences was estimated by counting differences between the human sequence (Supplementary Table S1) and the chimpanzee sequence (No- vember 2003 Assembly UCSC Browser [http://genome.ucsc. edu-cgi-bin/hgGateway]), initially at the overlapping length with the analyzed 2-kb human segment and subsequently extended to about 12 kb by adding 5 kb on each side to decrease variance in the d estimate. Both estimates largely agreed, and the latter is reported (Table 1). Prism v. 4.03 (GraphPad Software Inc., San Diego, CA), Excel (Microsoft, Redmond, WA), and Statistica