Full-length next-generation sequencing of HLA class I and II genes in a cohort from Thailand

The human leukocyte antigen (HLA) genes are highly variable and are known to play an important role in disease outcomes, including infectious diseases. Prior knowledge of HLA polymorphisms in a population usually forms the basis for an effective case-control study design. As a prelude to future disease association analyses, we report HLA class I and II diversity in 334 unrelated donors from a Dengue vaccine efficacy trial conducted in Thailand. Long-range PCR amplification of six HLA loci was performed on DNA extracted from saliva samples. HLA-A, -B, -C, -DPB1, -DQB1 and -DRB1 were genotyped using a next-generation sequencing method presented at the 17th International HLA and Immunogenetics Workshop. In total, we identified 201 HLA alleles, including 35 HLA-A, 57 HLA-B, 28 HLA-C, 24 HLA-DPB1, 21 HLA-DQB1 and 36 HLA-DRB1 alleles. Very common HLA alleles with frequencies greater than 10 percent were A111:01:01, A133:03:01, A124:02:01, B146:01:01, C107:02:01, C101:02:01, C108:01:01, DPB1105:01:01, DPB1113:01:01, DPB1104:01:01, DPB1102:01:02, DQB1103:01:01, DQB1105:02:01, DQB1103:03:02, DRB1112:02:01, DRB1109:01:02, and DRB1115:02:01. A novel HLA allele, B115:450, had a non-synonymous substitution and occurred in more than one donor. Population-based full-length NGS HLA typing is more conclusive and provides a sound foundation for exploring disease association in a given population.     

Next-generation sequencing of 11 HLA loci in a large dengue vaccine cohort from the Philippines

HLA genotyping by next-generation sequencing (NGS) has evolved with significant advancements in the last decade. Here we describe full-length HLA genotyping of 11 loci in 612 individuals comprising a dengue vaccine cohort from Cebu province in the Philippines. The multi-locus individual tagging NGS (MIT-NGS) method that we developed initially for genotyping 4–6 loci in one MiSeq run was expanded to 11 loci including HLA-A, B, C, DPA1, DPB1, DQA1, DQB1, DRB1, and DRB3/4/5. This change did not affect the overall coverage or depth of the sequencing reads. HLA alleles with frequencies greater than 10% were A*11:01:01, A*24:02:01, A*24:07:01, A*34:01:01, B*38:02:01, B*15:35, B*35:05:01, C*07:02:01, C*04:01:01, DPA1*02:02:02, DPB1*05:01:01, DPB1*01:01:01, DQA1*01:02:01, DQA1*06:01:01, DQB1*05:02:01, DQB1*03:01:01, DRB1*15:02:01, DRB1*12:02:01, DRB3*03:01:03, DRB4*01:03:01, and DRB5*01:01:01. Improvements in sequencing library preparation provide uniform and even coverage across all exons and introns. This has led to a marked reduction in allele imbalance and dropout. Furthermore, including more loci, such as DRB3/4/5, decreases cross-mapping and incorrect allele assignment at the DRB1 locus. The increased number of loci sequenced for each sample does not reduce the number of samples that can be multiplexed on a single MiSeq run and is therefore more cost-efficient. We believe that such improvements will help HLA genotyping by NGS to gain momentum over other conventional methods by increasing confidence in the calls.     

西北地区汉族人群HLA-A、-B、-DRB1基因座单倍型分析

目的 分析西北地区汉族群体HLA-A、-B和-DRB1基因座等位基因频率和HIA-A-B、B-DRB1和A-B-DRB1单倍型，获得单倍型频率数据。方法 采用序列特异性寡核苷酸探针反向斑点杂交技术对西北地区62个家系和101个无关个体HLA-A、-B和-DRB1基因座进行基因分型，分析HLA单倍型。结果 在西北地区汉族人群中检出15个HLA-A等位基因，28个HLA-B等位基因，13个HLA-DRB1等位基因，A02、A11、A24、B13、B15、1340、DRB1＊04、DRB1＊07、DRB1＊09和DRB1＊15基因频率较高（〉10％），A02（0．3244）、B13（0．1200）和DRB1＊15（0．1400）等位基因频率最高。分析得出HLA-A-B、B-DRB1、A-B-DRB1单倍型分别有122、147和278种，83种A-B-DRB1单倍型有至少两条以上相同的单倍型，占总单倍型数的18．44％（83／450）。A30-B13-DRB1＊07、A02-B46-DRB1＊09、A01-B37-DRB1＊10、A24-B15-DRB＊15、A02-B46-DRB1＊08、A33-B58-DRB1＊03是最常见的单倍型。结论 西北地区汉族群体HLA单倍型多态性较为丰富，等位基因频率和单倍型频率数据可用于骨髓移植供者的选择、法医学亲权鉴定以及人类学研究。     

Evaluation of Ion Torrent sequencing technology for rapid clinical human leucocyte antigen typing

The development of techniques to define the human leucocyte antigen (HLA) region has proven to be challenging due to its high level of polymorphism. Within a clinical laboratory, a technique for high‐resolution HLA typing, which is rapid and cost effective is essential. NGS has provided a rapid, high‐resolution HLA typing solution, which has reduced the number of HLA ambiguities seen with other typing methods. In this study, the One Lambda NXType NGS kit was tested on the Ion Torrent PGM platform. A total of 362 registry donors from four ethnic populations (Europeans, South Asians, Africans and Chinese) were NGS HLA typed across 9‐loci (HLA‐A, ‐B, ‐C, ‐DRB1,‐DRB345 ‐DQB1 and ‐DPB1). Concordance rates of 91%–98% were obtained (for HLA‐A, ‐B, ‐C, ‐DRB1, ‐DQB1 and ‐DPB1) when compared to historical PCR‐SSO HLA types, and the identification of uncommon alleles such as A*24:07:01 and C*04:82 were observed. A turnaround time of four days was achieved for typing 44 samples. However, some limitations were observed; primer locations did not allow all ambiguities to be resolved for HLA Class II where Exon I and IV amplification are needed (HLA‐DRB1*04:07:01/04:92, HLA‐DRB1*09:01:02/*09:21 and HLA‐DRB1*12:01:01/*12:10). This study has demonstrated high‐resolution typing by NGS can be achieved in an acceptable turnaround time for a clinical laboratory; however, the Ion Torrent workflow has some technical limitations that should be addressed.     

Distribution of human leucocyte antigen-A, -B and -DR alleles and haplotypes at high resolution in the population from Jiangsu province of China

The frequencies of human leucocyte antigen (HLA)-A, -B and -DRB1 alleles and haplotypes were statistically analysed among 3238 donors from Chinese Marrow Donor Program (CMDP) Jiangsu Branch. All donors were typed using polymerase chain reaction-sequence-based typing (PCR-SBT) method or polymerase chain reaction-reverse sequence-specific oligonucleotide probe (PCR-rSSOP) method. As a result, a total of 46 A, 85 B and 51 DRB1 alleles were found in Jiangsu population. The first three frequent alleles in HLA-A, -B and -DRB1 loci respectively were A*11:01(16.52%), A*24:02(15.10%) and A*02:01(13.02%); B*13:02(11.60%), B*46:01(8.89%) and B*58:01(7.12%); and DRB1*07:01(15.78%), DRB1*09:01(15.26%) and DRB1*15:01(9.76%). The top two frequent A-B-DRB1 haplotypes were A*30:01-B*13:02-DRB1*07:01(8.87%) and A*02:07-B*46:01-DRB1*09:01(2.79%); the top three A-B haplotypes were A*33:03-B*58:01-DRB1*03:01(2.59%), A*30:01-B*13:02(9.92%) and A*33:03-B*58:01(5.48%); the top two B-DRB1 haplotypes were B*13:02-DRB1*07:01(10.23%) and B*46:01-DRB1*09:01(4.61%); the top two A-DRB1 haplotypes were A*30:01-DRB1*07:01(8.96%) and A*33:03-DRB1*13:02(3.95%). These findings provided useful information in the study of genetics and anthropology in Chinese Han population. It also served as a basic guide for selection of future donors in CMDP Jiangsu Branch.     

Next generation sequencing to determine HLA class II genotypes in a cohort of hematopoietic cell transplant patients and donors

Current high-resolution HLA typing technologies frequently produce ambiguous results that mandate extended testing prior to reporting. Through multiplex sequencing of individual amplicons from many individuals at multiple loci, next generation sequencing (NGS) promises to eliminate heterozygote ambiguities and extend the breadth of genetic data acquired with little additional effort. We report here on assessment of a novel NGS HLA genotyping system for resequencing exons 2 and 3 of DRB1/B3/B4/B5, DQA1 and DQB1 and exon 2 of DPA1 and DPB1 on the MiSeq platform. In a cohort of 2605 hematopoietic cell transplant recipients and donors, NGS achieved 99.6% accuracy for DRB1 allele assignments and 99.5% for DQB1, compared to legacy genotypes generated pretransplant. NGS provided at least single 4-digit allele resolution for 97% of genotypes at DRB1 and 100% at DQB1. Overall, NGS typing identified 166 class II alleles, including 9 novel sequences with greater than 99% accuracy for DRB1 and DQB1 genotypes and elimination of diploid ambiguities through in-phase sequencing demonstrated the robust reliability of the NGS HLA genotyping reagents and analysis software employed in this study.     

HLA-A, -B,and -DRB1 genotyping and haplotype frequencies among Filipinos living in the National Capital Region of the Philippines

In this report, HLA polymorphisms (A, B, and DRB1 loci) were determined in 51 unrelated Filipinos. Molecular genotyping was carried out by sequence-based typing (Sanger sequencing). Data were analyzed by HLA-net GENE[RATE] tools. HLA-A, -B and -DRB1 genotype frequencies were at Hardy-Weinberg equilibrium. A*11, B*15, and DRB1*15 were the most frequent allele groups, while A*11-B*15-DRB1*15 was the most frequent HLA-A-B-DRB1 haplotype. HLA data are available in the Allele Frequencies Net Database (AFND: 3690) under the population name “Philippines National Capital Region”.     

Next generation sequencing of 11 HLA loci characterises a diverse UK cord blood bank

The introduction of next generation sequencing (NGS) for stem cell donor registry typing has contributed to faster identification of compatible stem cell donors. However, the successful search for a matched unrelated donor for some patient groups is still affected by their ethnicity.In this study, DNA samples from 714 National Health Service (NHS) Cord Blood Bank donors were typed for HLA-A, -B, -C, -DRB1, -DRB345, -DQA1, -DQB1, -DPA1 and -DPB1 by NGS. Analysis of the ethnic diversity showed a high level of diversity, with the cohort comprising of 62.3% European and 37.7% of either multi-ethnic or non-European donors, of which 12.3% were multi-ethnic. The HLA diversity was further confirmed using PyPop analysis, 405 distinct alleles were observed in the overall NHS-CBB cohort, of which 37 alleles are non-CWD, including A*31:14N, B*35:68:02, C*14:23 and DQA1*05:10. Furthermore, HLA-DQA1 and HLA-DPA1 analysis showed 12% and 10%, respectively, of the alleles currently submitted to IMGT, confirming further diversity of the NHS-CBB cohort.The application of 11 HLA loci resolution by NGS revealed a high level of diversity in the NHS-CBB cohort. The incorporation of this data coupled with ethnicity data could lead to improved donor selection, contributing to better clinical outcomes for patients.     

Distribution of human leukocyte antigen alleles and haplotypes in Oroqen and Ewenki nationality minority in Inner Mongolia Autonomous Region of China

The frequencies of the human leukocyte antigen alleles HLA-A,-B, DRB1 and the A-B, A-DRB1, B-DRB1, A-B-DRB1 haplotypes were investigated through means of PCR-based reverse line-strip sequence specific oligonucleotide hybridization on 108 Oroqen and 104 Ewenki nationality unrelated healthy individuals from the Inner Mongolia Autonomous Region of China. A total of thirteen different HLA-A alleles, 21 different HLA-B alleles and 13 different HLA-DRB1 alleles were detected in the Oroqen ethnic group and the most frequent HLA alleles found were A*24(35.65%), B*15(17.92%), and DRB1*09(17.59%), respectively. The common HLA-A-B-DRB1 haplotypes were A*24-B*40-DRB1*09(5.09%), A*24-B*48-DRB1*12(2.78%) and A*24-B*51-DRB1*04(2.78%); and the HLA-A*33-B*58, A*30-B*13, A*01-B*37, A*33-DRB1*03, A*01-DRB1*10, A*30-DRB1*07, B*37-DRB1*10, B*58-DRB1*03, B*38-DRB1*08, B*13-DRB1*07 were significant positive linkage disequilibrium in the Oroqen nationality group. In total, 14 different HLA-A alleles, 27 B alleles and 12 DRB1 alleles were found in Ewenki nationality group, and the most frequent HLA alleles found were A*24(24.49%), B*40(17.35%), and DRB1*04(14.80%), respectively. The common HLA-A-B-DRB1 haplotypes were A*33-B*58-DRB1*03(6.25%), A*01-B*51-DRB1*11(2.88%) and A*24-B*40-DRB1*09(2.88%); the HLA-A*33-B*58, A*29-B*44, A*03-B*52, A*33-DRB1*03, A*29-DRB1*07, A*24-DRB1*09, B*58-DRB1*03, B*08-DRB1*03, B*46-DRB1*09 were significant positive linkage disequilibrium in Ewenki nationality group. The distribution of HLA A,-B, DRB1, alleles haplotypes frequencies and phylogenetic tree indicated that the Oroqen and Ewenki population groups belongs to northern group of China, together as a group cluster.     

HLA-A, -B, and -DRB1 polymorphism defined by sequence-based typing of the Han population in Northern China

DNA typing for human leukocyte antigen (HLA)-A, -B and -DRB1 was performed using polymerase chain reaction-sequence-based typing method on 618 randomly selected healthy individuals of the Han population in Northern China. Allele frequencies and haplotypes were statistically analyzed. A total of 84 HLA-A alleles, 143 B alleles, and 122 DRB1 alleles were detected, and 853 A-B-DRB1 haplotypes, 473 A-B haplotypes, and 551 B-DRB1 haplotypes were statistically inferred. Statistical analysis of three-locus haplotypes showed that A*0207-B*4601-DRB1*0901 (3.06%) was the most predominant. Gene frequencies and haplotypic associations within HLA-A, -B, and -DRB1 loci were determined at a high-resolution (four digit) allelic level and should provide useful information in anthropology, bone marrow donor registry, legal medicine, and disease association studies.     

A single center study of protective and susceptible HLA alleles and haplotypes with end-stage renal disease in China

Chronic kidney disease (CKD) is becoming a global public health problem and usually cause End-Stage Renal Disease (ESRD) in the end of progression. To analyze the associations of HLA-A, -B, -C, -DRB1 and -DQB1 alleles at high resolution with ESRD in Jiangsu province of China, a total of 499 unrelated patients with ESRD from the First Affiliated Hospital with Nanjing Medical University and 1584 healthy controls from Jiangsu Branch of Chinese Marrow Donor Program (CMDP) were genotyped at HLA-A, -B, -C, -DRB1 and -DQB1 loci. Statistical analysis was applied to compare the differences of HLA allele frequencies between patients with ESRD and healthy controls. As results, no protective allele at A locus was found and the susceptible alleles were A*11:01 and A*31:01. At B locus, B*15:01, B*55:02 and B*39:05 emerged as susceptible alleles, whereas no protective allele was found. At C locus, C*06:02 and C*07:01 emerged as protective alleles and no susceptible allele was found. At DRB1 locus, six alleles including DRB1*03:01, DRB1*04:03, DRB1*04:04, DRB1*04:05, DRB1*11:01 and DRB1*12:02 emerged as susceptible alleles, while DRB1*15:01 emerged as a protective allele. At DQB1 locus, DQB1*02:01, DQB1*03:01, DQB1*03:02 and DQB1*04:01 emerged as susceptible alleles, while DQB1*06:02 and DQB1*06:09 emerged as protective alleles. Haplotype A*11:01-C*03:03-B*15:01-DRB1*11:01-DQB1*03:01 containing four susceptible alleles was regarded as the most susceptible haplotype. The susceptible alleles and haplotypes might be used as some important risk classification markers. Besides, in the consanguineous renal transplantation, it would be very beneficial for the long-term survival of renal transplant patients to avoid the susceptible alleles and haplotypes in selecting optimal donors.     

The distributions of HLA‐A,HLA‐B,HLA‐C,HLA‐DRB1 and HLA‐DQB1 allele and haplotype at high‐resolution level in Zhejiang Han population of China

The distributions of HLA allele and haplotype are variable in different ethnic populations and the data for some populations have been published. However, the data on HLA‐C and HLA‐DQB1 loci and the haplotype of HLA‐A, HLA‐B, HLA‐C, HLA‐DRB1 and HLA‐DQB1 loci at a high‐resolution level are limited in Zhejiang Han population, China. In this study, the frequencies of the HLA‐A, HLA‐B, HLA‐C, HLA‐DRB1 and HLA‐DQB1 loci and haplotypes were analysed among 3,548 volunteers from the Zhejiang Han population using polymerase chain reaction sequencing‐based typing method. Totals of 51 HLA‐A, 97 HLA‐B, 45 HLA‐C, 53 HLA‐DRB1 and 27 HLA‐DQB1 alleles were observed. The top three frequent alleles of HLA‐A, HLA‐B, HLA‐C, HLA‐DRB1 and HLA‐DQB1 loci were A*11:01 (23.83%), A*24:02 (17.16%), A*02:01 (11.36%); B*40:01 (14.08%), B*46:01 (12.20%), B*58:01 (8.50%); C*07:02 (18.25%), C*01:02:01G (18.15%), C*03:04 (9.88%); DRB1*09:01 (17.52%), DRB1*12:02 (10.57%), DRB1*15:01 (9.70%); DQB1*03:01 (22.63%), DQB1*03:03 (18.26%) and DQB1*06:01 (10.88%), respectively. A total of 141 HLA‐A‐C‐B‐DRB1‐DQB1 haplotypes with a frequency of ≥0.1% were found and the haplotypes with frequency greater than 3% were A*02:07‐C*01:02:01G‐B*46:01‐DRB1*09:01‐DQB1*03:03 (4.20%), A*33:03‐C*03:02‐B*58:01‐DRB1*03:01‐DQB1*02:01 (4.15%), A*30:01‐C*06:02‐B*13:02‐DRB1*07:01‐DQB1*02:02 (3.20%). The likelihood ratios test for the linkage disequilibrium of two loci haplotypes was revealed that the majority of the pairwise associations were statistically significant. The data presented in this study will be useful for searching unrelated HLA‐matched donor, planning donor registry and for anthropology studies in China.     

Study on the polymorphisms of HLA‐ABCDQB1DRB1 alleles and haplotypes in Hubei Han population of China

The present study aimed to analyse the frequencies of human leukocyte antigen HLA‐ABCDQB1 and HLA‐DRB1 alleles and haplotypes in a subset of 3,732 Han population from Hubei of China. All samples were typed in the HLA‐ABCDQB1 and HLA‐DRB1 loci using the sequence‐based typing method; subsequently, the HLA polymorphisms were analysed. A total of 47 HLA‐A, 89 HLA‐B, 43 HLA‐C, 49 HLA‐DRB1 and 24 HLA‐DQB1 alleles were identified in the Hubei Han population. The top three most frequent alleles in the HLA‐ABCDQB1 and HLA‐DRB1 were A*11:01 (0.2617), A*24:02 (0.1590), A*02:07 (0.1281); B*46:01 (0.1502), B*40:01 (0.1409) and B*58:01 (0.0616); C*01:02 (0.2023), C*07:02 (0.1691) and C*03:04 (0.1175); and DQB1*03:01 (0.2000), DQB1*03:03 (0.1900), DQB1*06:01 (0.1187); DRB1*09:01 (0.1790), DRB1*15:01 (0.1062) and DRB1*12:02 (0.0841), respectively. Meanwhile, the three most frequent two‐loci haplotypes were A*02:07‐C*01:02 (0.0929), B*46:01‐C*01:02 (0.1366) and DQB1*03:03‐DRB1*09:01 (0.1766). The three most frequent three‐loci haplotypes were A*02:07‐B*46:01‐C*01:02 (0.0883), B*46:01‐DQB1*03:03‐DRB1*09:01 (0.0808) and C*01:02‐DQB1*03:03‐DRB1*09:01 (0.0837). The three most frequent four‐loci haplotypes were A*02:07‐B*46:01‐C*01:02‐DQB1*03:03 (0.0494), B*46:01‐DRB1*09:01‐C*01:02‐DQB1*03:03 (0.0729) and A*02:07‐B*46:01‐DQB1*03:03‐DRB1*09:01 (0.0501). The most frequent five‐loci haplotype was A*02:07‐B*46:01‐C*01:02‐DQB1*03:03‐DRB1*09:01 (0.0487). Heat maps and multiple correspondence analysis based on the frequencies of HLA specificity indicated that the Hubei Han population might be described into Southern Chinese populations. Our results lay a certain foundation for future population studies, disease association studies and donor recruitment strategies.     

Polymorphism of human leukocyte antigen-A, -B, and -DRB1 in a Moroccan population from Casablanca: study of the allelic and the haplotypic frequencies

We have studied the distribution of HLA-A, -B and DRB1 alleles and haplotypes by sequence specific oligonucleotide amplification in a sample of 125 unrelated healthy Moroccan individuals from Casablanca in Morocco. The city of Casablanca is known of its big ethnic diversity, especially Arabs and Berbers. The most frequent alleles found were: HLA-A*02 (18.4%), -A*01 (11.2%), -A*03 (10.8%), -B*51 (8.06%),-B*44 (7.66%), -B*08 (6.85%), -DRB1*04 (15.98%), DRB1*03 and DRB1*07 (13.92%) and -DRB1*01 (10%). High frequency for five two-locus haplotypes was observed for A*03-B*51 (5%), A*02-DRB1*03 (5.5%), A*02-DRB1*04 and A*01-DRB1*04 (5%) and B*35-DRB1*04 (4%). No predominant haplotype was observed for HLA A-B-DRB1. Our results confirm and extend the current knowledge about genetic pattern of the Moroccan of Casablanca. This study will serve as a reference for further anthropological studies, as well as studies of HLA and disease associations in the Moroccan population.     

Allelic and haplotypic diversity of HLA-A, -B, -C, -DRB1, and -DQB1 genes in the Korean population

High-resolution human leukocyte antigen (HLA) typing exposes the unique patterns of HLA allele and haplotype frequencies in each population. In this study, HLA-A, -B, -C, -DRB1, and -DQB1 genotypes were analyzed in 485 apparently unrelated healthy Korean individuals. A total of 20 HLA-A, 43 HLA-B, 21 HLA-C, 31 HLA-DRB1, and 14 HLA-DQB1 alleles were identified. Eleven alleles (A*0201, A*1101, A*2402, A*3303, B*1501, Cw*0102, Cw*0302, Cw*0303, DQB1*0301, DQB1*0302, and DQB1*0303) were found in more than 10% of the population. In each serologic group, a maximum of three alleles were found with several exceptions (A2, B62, DR4, DR14, and DQ6). In each serologic group exhibiting multiple alleles, two major alleles were present at 62-96% (i.e. A*0201 and A*0206 comprise 85% of A2-positive alleles). Multiple-locus haplotypes estimated by the maximum likelihood method revealed 51 A-C, 43 C-B, 52 B-DRB1, 34 DRB1-DQB1, 48 A-C-B, 42 C-B-DRB1, 46 B-DRB1-DQB1, and 30 A-C-B-DRB1-DQB1 haplotypes with frequencies of more than 0.5%. In spite of their high polymorphism in B and DRB1, identification of relatively small numbers of two-locus (B-C and DRB1-DQB1) haplotypes suggested strong associations of those two loci, respectively. Five-locus haplotypes defined by high-resolution DNA typing correlated well with previously identified serology-based haplotypes in the population. The five most frequent haplotypes were: A*3303-Cw*1403-B*4403-DRB1*1302-DQB1*0604 (4.2%), A*3303-Cw*0701/6-B*4403-DRB1*0701-DQB1*0201/2 (3.0%), A*3303-Cw*0302-B*5801-DRB1*1302-DQB1*0609 (3.0%), A*2402-Cw*0702-B*0702-DRB1*0101-DQB1*0501 (2.9%), and A*3001-Cw*0602-B*1302-DRB1*0701-DQB1*0201/2 (2.7%). Several sets of allele level haplotypes that could not be discriminated by routine HLA-A, -B, and -DRB1 low-resolution typing originated from allelic diversity of A2, B61, DR4, and DR8 serologic groups. Information obtained in this study will be useful for medical and forensic applications as well as in anthropology.     

An integrated genotyping approach for HLA and other complex genetic systems

Clinical immunogenetics laboratories performing routine sequencing of human leukocyte antigen (HLA) genes in support of hematopoietic cell transplantation are motivated to upgrade to next-generation sequencing (NGS) technology by its potential for cost savings as well as testing accuracy and flexibility. While NGS machines are available and simple to operate, there are few systems available that provide comprehensive sample preparation and data analysis workflows to complete the process. We report on the development and testing of the Integrated Genotyping System (IGS), which has been designed to specifically address the challenges associated with the adoption of NGS in clinical laboratories. To validate the system for a variety of sample DNA sources, we have tested 336 DNA specimens from whole blood, dried blood spots, buccal swabs, and lymphoblastoid cell lines. HLA class I and class II genotypes were derived from amplicon sequencing of HLA-A, -B, -C for exons 1–7 and HLA-DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4, -DRB5 for exons 1–4. Additionally, to demonstrate the extensibility of the IGS to other genetic loci, KIR haplotyping of 93 samples was carried out in parallel with HLA typing using a workflow based on the HLA system. These results are discussed with respect to their applications in the clinical setting and consequent potential for advancing precision medicine.     

HLA-A, -B, -C, -DRB1 and -DQB1 alleles and haplotypes in the Kinh population in Vietnam

Allele and haplotype frequencies of the human leukocyte antigens (HLA) were studied in the Kinh Vietnamese population. We analyzed 170 unrelated healthy individuals. DNA-based HLA typing was performed using a microsphere-based array genotyping platform with sequence-specific oligonucleotide probes to distinguish HLA-A, -B, -C, -DRB1 and -DQB1 alleles. A total of 21 HLA-A, 37 HLA-B, 18 HLA-C, 25 HLA-DRB1, and 14 HLA-DQB1 alleles were identified. HLA-A*1101, A*2402, A*3303, B*1502, B*4601, Cw*0102, Cw*0702, Cw*0801, DRB1*1202, DQB1*0301, DQB1*0303, and DQB1*0501 were found with frequencies higher than 10%. Two representative haplotypes bearing two to five HLA loci were A*1101-B*1502 and A*3303-B*5801 for HLA-A-B; Cw*0801-B*1502 and Cw*0102-B*4601 for HLA-C-B; B*1502-DRB1*1202 and B*4601-DRB1*0901 for HLA-B-DRB1; DRB1*1202-DQB1*0301 and DRB1*0901-DQB1*0303 for HLA-DRB1-DQB1; A*1101-Cw*0801-B*1502 and A*3303-Cw*0302-B*5801 for HLA-A-C-B; A*1101-B*1502-DRB1*1202 and A*2901-B*0705-DRB1*1001 for HLA-A-B-DRB1, A*1101-Cw*0801-B*1502-DRB1*1202-DQB1*0301 and A*2901-Cw*1505-B*0705-DRB1*1001-DQB1*0501 for HLA-A-C-B-DRB1-DQB1. Allele distribution and haplotype analysis demonstrated that the Vietnamese population shares HLA patterns with southern Chinese, Thai, Javanese and Micronesians, while it also retains unique characteristics.     

应用SBT直接测序分型法研究中国南方汉族人群HLA五个基因座单倍型

目的 研究中国南方汉族人群HLA-A、B、Cw、DRBl、DQBl等位基因多态性及单倍型的分布特征.方法 应用聚合酶链反应-直接测序分型(polymerase chain reaction sequence-based typing,PCR-SBT)法对186名中国南方汉族健康人群HLA-A、B、Cw、DRBl、DQBl进行基因分型.结果 检出的HLA-A、B、Cw、DRBl、DQBl等位基因分别有28、49、24、29、20种.经统计分析A*0207-B*4601(10.81%),A*3303-B*5801(6.14%),B*4601-DRBl*0901(6.22%),B*4001*DRBl*0901(3.78%),DRBl*0901-DQBl*0303(12.16%)和DRBl*1202-DQBl*0301(8.38%)单倍型呈强连锁不平衡单倍型(RLF≥0.5,X<'2>>3.84,P<0.05);A*0207-B*4601-Cw*0102(10.75%),A*3303-B*5801-Cw*0302(5.14%),A*0207-B*4601-DR*0901(5.07%),A*3303-B*5801-DRBl*0301(2.96%),A*0207-B*4601-Cw*0102-DRBl*0901-DQBl*0303(4.87%)和A*1101-B*1301-Cw*0304-DRBl*1501-DQBl*0601(2.43%)单倍型分别是中国南方汉族人群常见单倍型.结论 中国南方汉族人群HLA 5个基因座单倍型分布具有高度的遗传多态性且有其自身分布特点.本研究获得的较完整的HLA 5个基因座单倍型分布数据,将为人类学、HLA疾病相关性和器官移植等研究提供遗传学参考数据.     

青海土族人群HLA-A、B、DRB1基因多态性分析

目的:分析青海土族人群人类白细胞抗原(Human leukocyte antigen,HLA)A、B、DRB1的基因多态性特点。方法:应用聚合酶链反应-直接测序分型(Polymerase chain reaction sequence-based typing,PCR-SBT)法对土族人群中47名健康无血缘关系的个体进行HLA-A、B、DRB1基因座高分辨分型。并将青海土族人群的HLA-DRB1基因与国内其它少数民族人群进行比较。结果:检出HLA-A、B、DRB1的基因型数和等位基因数分别为34、41、42和17、28、30。这3个基因座分布均符合Hardy-Weinberg平衡定律(P>0.05)。且HLA-DRB1基因座等位基因频率分布与蒙古族有相似之处,表现在基因频率较高的DRB1*04,DRB1*07,DRB1*09,DRB1*12等位基因。结论:青海土族人群具有独特的HLA-A、B、DRB1基因频率分布特征,且青海土族HLA-DRB1等位基因频率分布与蒙古族有相似之处。     

Super high resolution for single molecule-sequence-based typing of classical HLA loci at the 8-digit level using next generation sequencers

Current human leukocyte antigen (HLA) DNA typing methods such as the sequence-based typing (SBT) and sequence-specific oligonucleotide (SSO) methods generally yield ambiguous typing results because of oligonucleotide probe design limitations or phase ambiguity for HLA allele assignment. Here we describe the development and application of the super high-resolution single-molecule sequence-based typing (SS-SBT) of HLA loci at the 8-digit level using next generation sequencing (NGS). NGS which can determine an HLA allele sequence derived from a single DNA molecule is expected to solve the phase ambiguity problem. Eight classical HLA loci-specific polymerase chain reaction (PCR) primers were designed to amplify the entire gene sequences from the enhancer-promoter region to the 3' untranslated region. Phase ambiguities of HLA-A, -B, -C, -DRB1 and -DQB1 were completely resolved and unequivocally assigned without ambiguity to single HLA alleles. Therefore, the SS-SBT method described here is a superior and effective HLA DNA typing method to efficiently detect new HLA alleles and null alleles without ambiguity.