Cost-efficient high-throughput HLA typing by MiSeq amplicon sequencing.
Cost-efficient high-throughput HLA typing by MiSeq amplicon sequencing.
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DOI：
10.1186/1471-2164-15-63
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发表时间：
2014-01-24
期刊：
影响因子：
4.4
通讯作者：
Schmidt AH
中科院分区：
文献类型：
--
作者：
Lange V;Böhme I;Hofmann J;Lang K;Sauter J;Schöne B;Paul P;Albrecht V;Andreas JM;Baier DM;Nething J;Ehninger U;Schwarzelt C;Pingel J;Ehninger G;Schmidt AH
供受者HLA等位基因的高度匹配是非亲缘造血干细胞移植成功的重要前提。为了增加找到无关捐赠者的机会，登记处每年招募数十万志愿者。过去，许多资源有限的登记中心不得不在成本与分辨率和新招募供体的打字范围之间找到平衡。因此，我们利用NGS的最新改进来开发低成本、高分辨率HLA分型的工作流程。我们已经建立了一个简单的高通量HLA分型的三步工作流程：在Fluidigm Access Array微流控芯片上通过PCR扩增HLA-A、-B、-C、-DRB 1、-DQB 1和-DPB 1的外显子2和3。在PCR期间直接掺入Illumina测序衔接子和样品特异性标签。在合并和净化后，在单个Illumina MiSeq运行中对384个样品进行测序。我们开发了“neXtype”用于简化数据分析和HLA等位基因分配。用1140份在6个位点分型的样本验证了工作流程。所有neX分型结果与桑格序列一致，表明超过6000个HLA位点的无错误分型。目前常规操作的能力为每周12，000个样本。所提出的工作流程被证明是高通量HLA分型的桑格测序的一种具有成本效益的替代方案。尽管注重成本效益，但分辨率超过了当前用于供体登记的桑格分型标准。
A close match of the HLA alleles between donor and recipient is an important prerequisite for successful unrelated hematopoietic stem cell transplantation. To increase the chances of finding an unrelated donor, registries recruit many hundred thousands of volunteers each year. Many registries with limited resources have had to find a trade-off between cost and resolution and extent of typing for newly recruited donors in the past. Therefore, we have taken advantage of recent improvements in NGS to develop a workflow for low-cost, high-resolution HLA typing. We have established a straightforward three-step workflow for high-throughput HLA typing: Exons 2 and 3 of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 are amplified by PCR on Fluidigm Access Array microfluidic chips. Illumina sequencing adapters and sample specific tags are directly incorporated during PCR. Upon pooling and cleanup, 384 samples are sequenced in a single Illumina MiSeq run. We developed “neXtype” for streamlined data analysis and HLA allele assignment. The workflow was validated with 1140 samples typed at 6 loci. All neXtype results were concordant with the Sanger sequences, demonstrating error-free typing of more than 6000 HLA loci. Current capacity in routine operation is 12,000 samples per week. The workflow presented proved to be a cost-efficient alternative to Sanger sequencing for high-throughput HLA typing. Despite the focus on cost efficiency, resolution exceeds the current standards of Sanger typing for donor registration.
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影响因子：
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作者：
Bentley G;Higuchi R;Hoglund B;Goodridge D;Sayer D;Trachtenberg EA;Erlich HA
通讯作者：
Erlich HA
影响因子：
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Robinson J;Halliwell JA;McWilliam H;Lopez R;Parham P;Marsh SG
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Marsh SG
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作者：
Schmidt, Alexander H.;Baier, Daniel;Rutt, Claudia
通讯作者：
Rutt, Claudia
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Xiao, Y.;Lazaro, A. M.;Hurley, C. K.
通讯作者：
Hurley, C. K.
影响因子：
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作者：
Marsh SG;Albert ED;Bodmer WF;Bontrop RE;Dupont B;Erlich HA;Fernández-Viña M;Geraghty DE;Holdsworth R;Hurley CK;Lau M;Lee KW;Mach B;Maiers M;Mayr WR;Müller CR;Parham P;Petersdorf EW;Sasazuki T;Strominger JL;Svejgaard A;Terasaki PI;Tiercy JM;Trowsdale J
通讯作者：
Trowsdale J