Genomic characterization of MICA gene using multiple next generation sequencing platforms: A validation study.
Genomic characterization of MICA gene using multiple next generation sequencing platforms: A validation study.
复制标题
使用多个下一代测序平台对 MICA 基因进行基因组表征：验证研究
作者：
Zou Y;Duke JL;Ferriola D;Luo Q;Wasserman J;Mosbruger TL;Luo W;Cai L;Zou K;Tairis N;Damianos G;Pagkrati I;Kukuruga D;Huang Y;Monos DS
我们已经开发了一个关于通过下一代测序（NGS）对云母基因进行基因组表征的方案。扩增子包括基因的全长，约为13 kb。本研究共纳入156份样本。这些样本中有97份先前在云母通过传统方法（桑格或序列特异性寡核苷酸）进行了表征，并用于评价测定的准确度、精密度、特异性和灵敏度。另外59份来自美国的未知种族志愿者的DNA样本仅通过NGS进行基因分型。选择含有不同等位基因组的样品。我们的NGS方法包括在Illumina MiSeq平台上的第一轮测序和在MinION平台上由Oxford Nanopore Technology（ONT）进行的第二轮测序，对选定的样品进行测序，目的是表征新的等位基因或在多个多态性之间设置相位以解决歧义或生成在IMGT/HLA数据库中仅部分报告的等位基因的完整序列。用ONT测序的每个等位基因都产生了完整的共有序列，从云母基因的5′非翻译区（UTR）延伸到3′ UTR。将32个云母序列提交至IMGT/HLA数据库，包括新等位基因或填补已报告等位基因的空位（外显子、内含子和/或UTR）。与这些样品的表征相关的一些挑战进行了讨论。
We have developed a protocol regarding the genomic characterization of the MICA gene by next generation sequencing (NGS). The amplicon includes the full length of the gene and is about 13 kb. A total of 156 samples were included in the study. Ninety‐seven of these samples were previously characterized at MICA by legacy methods (Sanger or sequence specific oligonucleotide) and were used to evaluate the accuracy, precision, specificity, and sensitivity of the assay. An additional 59 DNA samples of unknown ethnicity volunteers from the United States were only genotyped by NGS. Samples were chosen to contain a diverse set of alleles. Our NGS approach included a first round of sequencing on the Illumina MiSeq platform and a second round of sequencing on the MinION platform by Oxford Nanopore Technology (ONT), on selected samples for the purpose of either characterizing new alleles or setting phase among multiple polymorphisms to resolve ambiguities or generate complete sequence for alleles that were only partially reported in the IMGT/HLA database. Complete consensus sequences were generated for every allele sequenced with ONT, extending from the 5′ untranslated region (UTR) to the 3′ UTR of the MICA gene. Thirty‐two MICA sequences were submitted to the IMGT/HLA database including either new alleles or filling up the gaps (exonic, intronic and/or UTRs) of already reported alleles. Some of the challenges associated with the characterization of these samples are discussed.
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DOI：
10.1016/j.bbmt.2016.11.021
发表时间：
2017-03
期刊：
Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation
影响因子：
--
作者：
Askar M;Sobecks R;Wang T;Haagenson M;Majhail N;Madbouly A;Thomas D;Zhang A;Fleischhauer K;Hsu K;Verneris M;Lee SJ;Spellman SR;Fernández-Viña M
通讯作者：
Fernández-Viña M
影响因子：
12.3
作者：
Rang FJ;Kloosterman WP;de Ridder J
通讯作者：
de Ridder J
影响因子：
56.9
作者：
Bauer, S;Groh, V;Spies, T
通讯作者：
Spies, T
影响因子：
--
作者：
Romphruk, AV;Naruse, TK;Inoko, H
通讯作者：
Inoko, H
DOI：
10.1073/pnas.91.14.6259
发表时间：
1994-07-05
影响因子：
11.1
作者：
BAHRAM, S;BRESNAHAN, M;SPIES, T
通讯作者：
SPIES, T