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Title: | 應用次世代定序技術鑑定人類白血球表面抗原基因型 HLA Typing Using Capture-based Next-generation Sequencing |
Authors: | Sheng-Kai Lai 賴勝凱 |
Advisor: | 陳沛隆 |
Keyword: | 人類白血球抗原,探針捕獲,次世代定序, Human leucocyte antigen,HLA,Probe-capture,NGS, |
Publication Year : | 2018 |
Degree: | 碩士 |
Abstract: | 人類白血球抗原(HLA)對於器官移植與免疫相關疾病扮演相當重要的角色,傳統的人類白血球抗原型別鑑定,通常使用桑格定序法或序列特異性寡核苷酸分型法,常常因為定序無法區分單套型以及特異點位不在定序範圍內而給出有歧異的結果。近年來,國外的研究團隊紛紛利用次世代定序,作為HLA基因型鑑定的工具。目前市面上的次世代定序平台,多以長片段聚合酶鏈鎖反應產物為基礎進行定序,因此聚合酶鏈鎖反應的缺點也會在結果上呈現,例如:非專一性放大、等位基因丟失 (allele drop out)、聚合酶鏈鎖反應錯誤 (PCR error)…等等。使用探針捕獲的方式可解決上述的問題。
本研究建立以探針捕獲為基本的次世代定序方法利用在人類白血球抗原型別鑑定上,探針針對第一型人類白血球抗原 (HLA-A, B, C)、第二型人類白血球抗原 (HLA-DRB1, DPA1, DPB1, DQA1, DQB1, DRB3, DRB4, DRB5) 以及MICA的外顯子2到4進行設計。第一階段以10個帶有台灣常見型別的檢體進行測試,實驗結果與先前利用定序分型的結果相符合。緊接著以國際組織相容協會 (International Histocompatibility Working Group, IHWG) 的 85 個標準品來進行測試,結果顯示99%的結果相符合,其他的1%檢體以傳統定序法確認,包括1個HLA-A、2個HLA-DPA1、9個HLA-DPB1、4個HLA-DQA1、4個DQB1以及1個DRB1,發現結果與本研究相同,顯示IHWG的結果可能因為種種因素有誤。異型合子的兩個基因座讀深比例平均大約為40%-60%,顯示本研究方法對於異型合子的檢體不會產生傳統方法容易出現非專一性放大、等位基因丟失的問題,並且能夠解決因無法區分單套型而造成的歧異結果。 Human leucocyte antigens (HLA) are critical in organ transplantation and immune-related diseases. Traditional HLA typing methods, either by sequence-based typing (SBT) or sequence-specific oligonucleotide (SSO), might report ambiguous typing results, which are caused by the phasing problem and the polymorphic sites outside the sequencing regions. Recent next-generation sequencing (NGS)-based methods rely on long-range PCR, which might suffer from allele drop-out. Using probe capture-based NGS may solve the mentioned obstacles. In the study, we established a probe capture-based NGS method for HLA typing. Probe sets for class-I HLA genes (HLA-A, B, C), class-II HLA genes (HLA-DRB1, DPA1, DPB1, DQA1, DQB1, DRB3, DRB4, DRB5) and MICA gene covering exon 2 to exon 4 were designed. Ten DNA samples carrying the most common HLA types in Taiwanese were used as the stage one validation for this method. The results were 100% concordant to those from the SBT method. We then proceeded to test the 85 samples from The International HLA Reference Standards from International Histocompatibility Working Group (IHWG). An average of 99% results were consistent with the results from IHWG. The remaining 1% results were inconsistent, including 1 A allele, 2 DPA1 alleles, 9 DPB1 alleles, 4 DQA1 alleles, 4 DQB1 alleles and 1 DRB1 allele; we later proved that those results from IHWG were wrong. The ratio of two alleles read depth in heterozygous loci were 40%-60% in average. Furthermore, our data showed that the established method could resolve ambiguous HLA types due to phasing problems. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70519 |
DOI: | 10.6342/NTU201803072 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 分子醫學研究所 |
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ntu-107-1.pdf Restricted Access | 4.44 MB | Adobe PDF |
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