Defining kir and hla class I genotypes at Highest Resolution via High-Throughput Sequencing
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haplotypes in the cell panel ( Figure 6
and Table S3
). Consis- tent with our results are KIR genotyping data obtained pre- viously from the IHWG cells, which achieved a limited discrimination of alleles. 6,59 These analyses demonstrate that PING accurately processes high-throughput sequence data to give accurate high-resolution KIR genotypes. The high-resolution KIR genotypes of the IHWG cell panel have been compiled ( Table S3 ), providing a resource for future investigation. Validation of the High-Resolution HLA Class I Capture Method Because KIR and HLA class I glycoproteins are function- ally interacting receptors and ligands, the capture/NGS method was designed to capture and analyze the two gene families in the same reaction ( Material and Methods ). To validate the HLA class I sequences obtained by this method, we first analyzed the panel of 97 IHWG cell lines. In previous studies, 38–40 including high-density whole- genome SNP analysis, 43 90 of the IHGW cells were judged to be homozygous for HLA-A, HLA-B, and HLA-C, and five of the other seven lines were found to be homozygous for two of the three HLA class I genes. Our high- throughput sequencing results are completely concordant with the genotypes previously determined by conven- tional methods 15 (
A). In the course of validation, we defined two novel HLA-C alleles that encode distinctive proteins ( Table S4
B). Because our method gives full-length genomic sequences, including introns and flanking re- gions, all HLA-A, HLA-B, and HLA-C alleles of the IHWG cells are now defined at a much higher resolution (‘‘four field’’; see Appendix A ) than previously achieved. The IHWG cells represent an unusual, and highly selected, sampling of the human population because they are HLA homozygous, and many of them derive from consanguineous individuals. To extend the study to heterozygous individuals, we applied the capture/NGS method to 30 West African family trios from Mali (sample set 2,
Material and Methods ) and 188 individuals selected at random from a panel of healthy Europeans (sample set 3). The capture/NGS method achieved full coverage of the HLA class I genes, and an example of the result ob- tained from one individual is shown in Figure 7 A. In the Africans, 22 HLA-A, 30 HLA-B, and 15 HLA-C alleles were identified. These allele sequences agreed completely with HLA class I genotypes we determined by standard probe- based and Sanger sequencing methods. They were also consistent with the observed segregation of HLA-A, HLA-B, and HLA-C alleles within the family trios ( Tables S4C–S4
D). In total, 170 distinct HLA class I alleles were identified in the three validation sets (IHWG cell lines, Af- rican trios, and Europeans). These 170 alleles include 62 of the 69 fundamental allele types defined by the first two digits of the HLA nomenclature ( Figure 7
B) and thus cover most, if not all, of the breadth of HLA class I allelic diver- sity. 21
alleles can be captured and sequenced by our method. In support of this thesis, the capture/NGS method robustly detects and sequences B*73:01 (E.A., unpublished data), a unique, archaic allele that has by far the most divergent KIR 1. SP0010 2. CB6B 3. E481324 4. LZL 3DL3
*00206 *00206
*01403 *00301
*00206 *01501
*00802 *00402
2DS2 *001
*001 *001
2DL2/3 L3*001
L3*001 L2*001
L2*003 L3*001
L3*001 L3*001
L2*003 2DL5
B*002 2DS3/5
3*00103 2DP1
*00203 *00203
*012 *00203
*00204 *002
2DL1 *00302
*00302 *00401
*00302 *00302
*00302 3DP1
*00302 *00302
*00301 *009
*00302 *00302
*00302 3DP1b
*001 2DL4
*011 *011
*00501 *00501
*00103 *00802
*00102 2DL4b
*00501 3DL1/S1
L1*00501 L1*00501 S1*013
S1*013 L1*002
L1*00401 L1*01502
3DL1/S1b 3DS1*013
2DL5 A*001
A*005 B*010
2DS3/5 5*002
3*002 3*002
2DS1 *002
*002 *002
2DS4 *010
*010 *00101
*006 *00101
3DL2 *00103
*00103 *00701
*00701 *00103
*00501 *00201
*00701 Figure 6. High-Resolution KIR Allele and Copy-Number Genotypes of 97 IHWG Cells Four examples of high-resolution allele and copy-number genotypes of KIR. Individual 1 (SP0010) is homozygous for the KIR A haplo- type. Individual 2 (CB6B) has two different B haplotypes. Individual 3 (E481324) has a duplication of three loci (in blue shading: denoted as 3DP1b, 2DL4b, and 3DL1/S1b). Individual 4 (LZL) has a deletion of the central segment of the KIR haplotype (red). Yellow shading denotes alleles that were identified in the current study. The full genotypes for each IHWG cell are given in Table S3
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The American Journal of Human Genetics 99, 375–391, August 4, 2016 Yüklə 448,46 Kb. Dostları ilə paylaş:
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