Yin Yuxin, Lan James H, Nguyen David, Valenzuela Nicole, Takemura Ping, Bolon Yung-Tsi, Springer Brianna, Saito Katsuyuki, Zheng Ying, Hague Tim, Pasztor Agnes, Horvath Gyorgy, Rigo Krisztina, Reed Elaine F, Zhang Qiuheng
UCLA Immunogenetics Center, Department of Pathology & Laboratory Medicine, Los Angeles, CA, United States of America.
University of British Columbia Clinician Investigator Program, Vancouver, BC, Canada.
PLoS One. 2016 Oct 31;11(10):e0165810. doi: 10.1371/journal.pone.0165810. eCollection 2016.
Unambiguous HLA typing is important in hematopoietic stem cell transplantation (HSCT), HLA disease association studies, and solid organ transplantation. However, current molecular typing methods only interrogate the antigen recognition site (ARS) of HLA genes, resulting in many cis-trans ambiguities that require additional typing methods to resolve. Here we report high-resolution HLA typing of 10,063 National Marrow Donor Program (NMDP) registry donors using long-range PCR by next generation sequencing (NGS) approach on buccal swab DNA.
Multiplex long-range PCR primers amplified the full-length of HLA class I genes (A, B, C) from promotor to 3' UTR. Class II genes (DRB1, DQB1) were amplified from exon 2 through part of exon 4. PCR amplicons were pooled and sheared using Covaris fragmentation. Library preparation was performed using the Illumina TruSeq Nano kit on the Beckman FX automated platform. Each sample was tagged with a unique barcode, followed by 2×250 bp paired-end sequencing on the Illumina MiSeq. HLA typing was assigned using Omixon Twin software that combines two independent computational algorithms to ensure high confidence in allele calling. Consensus sequence and typing results were reported in Histoimmunogenetics Markup Language (HML) format. All homozygous alleles were confirmed by Luminex SSO typing and exon novelties were confirmed by Sanger sequencing.
Using this automated workflow, over 10,063 NMDP registry donors were successfully typed under high-resolution by NGS. Despite known challenges of nucleic acid degradation and low DNA concentration commonly associated with buccal-based specimens, 97.8% of samples were successfully amplified using long-range PCR. Among these, 98.2% were successfully reported by NGS, with an accuracy rate of 99.84% in an independent blind Quality Control audit performed by the NDMP. In this study, NGS-HLA typing identified 23 null alleles (0.023%), 92 rare alleles (0.091%) and 42 exon novelties (0.042%).
Long-range, unambiguous HLA genotyping is achievable on clinical buccal swab-extracted DNA. Importantly, full-length gene sequencing and the ability to curate full sequence data will permit future interrogation of the impact of introns, expanded exons, and other gene regulatory sequences on clinical outcomes in transplantation.
明确的人类白细胞抗原（HLA）分型在造血干细胞移植（HSCT）、HLA疾病关联研究及实体器官移植中至关重要。然而，当前的分子分型方法仅检测HLA基因的抗原识别位点（ARS），导致许多顺反式模糊性，需要额外的分型方法来解决。在此，我们报告了采用下一代测序（NGS）方法，通过长程PCR对10,063名国家骨髓捐献计划（NMDP）登记捐献者的口腔拭子DNA进行高分辨率HLA分型。
多重长程PCR引物从启动子到3'非翻译区（UTR）扩增HLA I类基因（A、B、C）的全长。II类基因（DRB1、DQB1）从外显子2扩增至外显子4的部分区域。PCR扩增产物混合后，使用Covaris片段化仪进行片段化处理。使用Illumina TruSeq Nano试剂盒在Beckman FX自动化平台上进行文库制备。每个样本用独特的条形码标记，随后在Illumina MiSeq上进行2×250 bp双端测序。使用Omixon Twin软件进行HLA分型，该软件结合了两种独立的计算算法，以确保等位基因分型的高可信度。以组织免疫遗传学标记语言（HML）格式报告一致性序列和分型结果。所有纯合等位基因通过Luminex SSO分型进行确认，外显子新奇性通过Sanger测序进行确认。
使用这种自动化流程，通过NGS成功对超过10,063名NMDP登记捐献者进行了高分辨率分型。尽管已知基于口腔样本的核酸降解和低DNA浓度的挑战，但97.8%的样本通过长程PCR成功扩增。其中，98.2%的样本通过NGS成功报告，在由NDMP进行的独立盲法质量控制审核中的准确率为99.84%。在本研究中，NGS-HLA分型鉴定出23个无效等位基因（0.023%）、92个罕见等位基因（0.091%）和42个外显子新奇性（0.042%）。
在临床口腔拭子提取的DNA上可实现长程、明确的HLA基因分型。重要的是，全长基因测序以及整理完整序列数据的能力将允许未来探究内含子、扩展外显子和其他基因调控序列对移植临床结果的影响。
