Supporting information for Bamshad et al. (2002) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.162046399

 

Supporting Appendix

Table 3. Frequencies of minor alleles found in world and National Institutes of Health (NIH) panels

Nucleotide position

Major allele, minor allele

Old World panel (n = 224)

NIH panel (n = 180)

–2852

A,G

0.295

0.273

–2838

A,G

0.004

0.006

–2733

A,G

0.036

0.045

–2554

G,T

0.397

0.386

–2459

G,A

0.442

0.500

–2454*

G,A

0.004

0.000

–2256*

A,G

0.009

0.000

–2150*

A,G

0.005

0.000

–2135

T,C

0.428

0.500

–2132

C,T

0.032

0.040

–2086

A,G

0.355

0.347

–1915*

G,A

0.005

0.000

–1835

C,T

0.149

0.256

*Polymorphism present in Old World panel, absent in NIH panel.

 

Populations Sampled

The populations sampled included 31 Africans (3 Biaka Pygmies, 7 Mbuti Pygmies, 3 Alur, 3 Nande, 3 Hema, 1 Kenyan, 2 Nigerians, 3 Sotho, 3 Nguni, and 3 San), 27 non-Indian Asians (3 Cambodians, 5 Han Chinese, 6 Japanese, 3 aboriginal Malayans, 3 Vietnamese, 2 Mongolians, 1 Nicobarese, 3 Daghestanis, and 1 Asian of mixed Asian ancestry), 24 Europeans (4 North Europeans, 5 French, 4 Italians, 5 Poles, and 6 Finns), and 30 South Indians (3 Brahmin, 3 Kshatriya, 2 Kapu, 3 Yadava, 3 Mala, 3 Madiga, 3 Relli, 3 Irula, 3 Khonda Dora, 1 Santal, and 3 Chenchu).

Sequencing Methods

The entire region was amplified using CCR5L2-CCAAACTGTGACCCTTTCC and CCR5R2-CAGATGTCACCAACCGC. Sequencing was performed by using the following internal primers: CCR5A-CGGGCTTTTCTCACTGG, CCR5B-AACTATGGGCTCACGGG, CCR5D-CCGTAAATAAACCTTCAGACCAG, CCR5E-CAGGGCTTTTCAACAGTAAGG, CCR5F-TTCTTTTGAAGGAGGGTGGAG, CCR5H-GCCTTACTGTTGAAAAGCC, and CCR5I-GGTTAATGTGAAGTCCAGG. PCR amplification was carried out in 50-ml reaction volumes by using Expand Long Template PCR System (Roche). Each reaction contained 50 ng of genomic DNA, 350 mM dNTPs, 0.2 mM of each PCR primer, 1´ reaction buffer no. 2, and 2.6 units of Taq/Pwo DNA polymerase mix. Cycling conditions included an initial denaturation at 94°C for 2 min, 10 cycles of 94°C for 10 sec, 55°C for 10 sec, 68°C for 2 min; followed by 15 cycles of 94°C for 10 sec, 55°C for 10 sec, 68°C for 2 min +20 sec per cycle. Residual primers and dNTPs were removed from PCR products with a Millipore glass fiber filter. The sequence-ready templates were eluted in 70 ml of sterile H2O. Five microliters of each template was aliquoted to 12 wells of a 384-well sequence dish and evaporated to dryness in a speed-vac. Cycle sequencing was carried out in 5-ml reaction volumes by using ABI BigDye Terminator chemistry. Cycling conditions included an initial denaturation at 96°C for 30 sec; followed by 46 cycles of 96°C for 10 sec, 50°C for 5 sec, 60°C for 4 min. Upon completion of cycle sequencing, 20 ml of 62.5% ETOH/1 M KOAc, pH 4.5 was added to each reaction, and the sequence plates were centrifuged at 4,000 rpm in an Eppendorf centrifuge at 4°C for 45 min. The samples were resuspended in 15 ml of sterile H2O and electrophoresed on an ABI 3700 DNA analyzer prepared with POP-5 capillary gel matrix.

Comparison to Nonhuman Primates

The chimpanzees and gorilla were monomorphic at each polymorphic human site. This permitted the assignment of the ancestral vs. derived state for each human single nucleotide polymorphism. Comparison of the human and great ape sequences revealed that the common allele at each of the 13 polymorphic sites in the Old World panel was shared by the chimpanzees and gorilla. The bonobo and chimpanzees differed from one another at 4 fixed sites. The chimpanzees were monomorphic for a 2-bp (i.e., CA) deletion in intron 1, and 2 common chimpanzees had a 7-bp deletion (TTTATTC) in intron 1. Neither of these deletions is present in either bonobo or human.