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Rapid, highly accurate and cost-effective open-source simultaneous complete HLA typing and phasing of class I and II alleles using nanopore sequencing.
HLA ( IF 5.9 ) Pub Date : 2020-07-13 , DOI: 10.1111/tan.13926
Joanne D Stockton 1 , Thomas Nieto 1 , Elizabeth Wroe 2 , Anthony Poles 2 , Nicholas Inston 3 , David Briggs 2 , Andrew D Beggs 1
Affiliation  

Accurate rapid genotyping of the genes within the HLA region presents many difficulties because of the complexity of this region. Here we present the results of our proof of concept nanopore‐based long read polymerase chain reaction (PCR) solution for HLA genotyping. For 15 HLA anthropology‐based samples and 13 NHS Blood and Transplant derived samples 40 ng of genomic DNA underwent long‐range PCR for class I and II HLA alleles. Pooled PCR products were sequenced on the Oxford Nanopore MinIoON R9.4.1 flow cell. Sequenced reads had HLA genotype assigned with HLA‐LA. Called genotypes were compared with reference derived from a combination of short‐read next‐generation sequencing, Sanger sequence and/or single‐site polymorphism (SSP) typing. For concordance, accuracy was 100%, 98.4%, 97.5% and 95.1% for the first, second, third and fourth fields, respectively, to four field accuracy where it was available, otherwise three field in 28 samples for class I calls and 17 samples for class II calls. Phasing of maternal and paternal alleles, as well as phasing based identification of runs of homozygosity, was shown successfully. Time for assay run was 8 hours and the reconstruction of HLA typing data was 15 minutes. Assay cost was £55 ($80USD)/sample. We have developed a rapid and cost‐effective long‐range PCR and nanopore sequencing‐based assay that can genotype the genes within HLA region to up to four field accuracy, identify runs of homozygosity in HLA, reconstruct maternal and paternal haplotypes and can be scaled from multi‐sample runs to a single sample.

中文翻译:


使用纳米孔测序对 I 类和 II 类等位基因进行快速、高度准确且经济高效的开源同步完整 HLA 分型和定相。



由于 HLA 区域的复杂性,对 HLA 区域内的基因进行准确快速的基因分型存在许多困难。在这里,我们展示了用于 HLA 基因分型的基于纳米孔的长读长聚合酶链反应 (PCR) 解决方案的概念验证结果。对于 15 个基于 HLA 人类学的样本和 13 个 NHS 血液和移植来源的样本,对 40 ng 基因组 DNA 进行了 I 类和 II 类 HLA 等位基因的长程 PCR。合并的 PCR 产物在 Oxford Nanopore MinIoON R9.4.1 流动池上进行测序。测序读数具有 HLA-LA 指定的 HLA 基因型。将所谓的基因型与短读长下一代测序、桑格序列和/或单位点多态性 (SSP) 分型组合得出的参考进行比较。为了一致性,第一、第二、第三和第四场的准确度分别为 100%、98.4%、97.5% 和 95.1%,在可用的情况下达到四个场的准确度,否则 I 类调用的 28 个样本中的三个场和 17 个II 类呼叫的样本。母本和父本等位基因的定相以及基于定相的纯合性运行鉴定均已成功展示。检测运行时间为 8 小时,HLA 分型数据重建时间为 15 分钟。检测成本为 55 英镑(80 美元)/样本。我们开发了一种快速且经济高效的基于长程 PCR 和纳米孔测序的检测方法,可以对 HLA 区域内的基因进行基因分型,精度高达 4 个字段,识别 HLA 中的纯合性运行,重建母本和父本单倍型,并且可以扩展从多样本运行到单样本运行。
更新日期:2020-07-13
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