High throughput HLA genotyping using 454 sequencing and the Fluidigm Access Arrayâ„¢ system for simplified amplicon library preparation
High throughput HLA genotyping using 454 sequencing and the Fluidigm Access Arrayâ„¢ system for simplified amplicon library preparation
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DOI：
10.1111/tan.12071
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发表时间：
2013-03-01
期刊：
影响因子：
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通讯作者：
Erlich, H. A.
中科院分区：
文献类型：
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作者：
Moonsamy, P. V.;Williams, T.;Erlich, H. A.
人类白细胞抗原（HLA） I类和II类位点是人类基因组中多态性最多的基因；区分成千上万的HLA等位基因是一项挑战。利用454基因组序列（GS） FLX系统和CONEXIO ASSIGN ATF 454软件进行下一代外显子扩增子测序，为8个I类和II类基因座提供高分辨率、高通量的HLA基因分型。非亲属骨髓供体登记中潜在供者的HLA分型通常使用这些基因座的一个子集，具有高样本吞吐量和低样本成本。Fluidigm访问阵列系统能够将48种不同的多重标识符（MIDs）与48个基因组DNA样本相对应，在微流控装置中具有多达48种不同的引物对，可产生2304种平行聚合酶链反应（pcr）。使用最小体积的试剂。在基因组PCR过程中，在这个4引物系统中，包含MID和454接头序列的外部引物被整合到由内部HLA靶特异性引物产生的扩增子中，每个引物在正引物和反向引物的5'端都包含一个共同的序列标签。所得扩增子池在454 Life Sciences GS FLX系统上进行乳状PCR和克隆测序，然后用CONEXIO软件进行基因分型。我们使用8对引物（覆盖HLA-A、B和C的外显子2和3，以及DRB1、3/4/5和DQB1的外显子2）和96个MIDs，在一次GS FLX运行中对192个样本进行了基因分型，与已知基因型100%一致。每个扩增子平均有166个reads。我们还对96个样本进行了高分辨率的基因分型（14对引物覆盖了DRB1、3/4/5、DQA1、DQB1、DPB1和DQB1外显子2的I类位点的2、3和4外显子和2外显子），平均每个扩增子有173个序列读取。
The human leukocyte antigen (HLA) class I and class II loci are the most polymorphic genes in the human genome; distinguishing the thousands of HLA alleles is challenging. Next generation sequencing of exonic amplicons with the 454 genome sequence (GS) FLX System and CONEXIO ASSIGN ATF 454 software provides high resolution, high throughput HLA genotyping for eight class I and class II loci. HLA typing of potential donors for unrelated bone marrow donor registries typically uses a subset of these loci at high sample throughput and low cost per sample. The Fluidigm Access Array System enables the incorporation of 48 different multiplex identifiers (MIDs) corresponding to 48 genomic DNA samples with up to 48 different primer pairs in a microfluidic device generating 2304 parallel polymerase chain reactions (PCRs). Minimal volumes of reagents are used. During genomic PCR, in this 4-primer system, the outer set of primers containing the MID and the 454 adaptor sequences are incorporated into an amplicon generated by the inner HLA target-specific primers each containing a common sequence tag at the 5' end of the forward and reverse primers. Pools of the resulting amplicons are used for emulsion PCR and clonal sequencing on the 454 Life Sciences GS FLX System, followed by genotyping with CONEXIO software. We have genotyped 192 samples with 100% concordance to known genotypes using 8 primer pairs (covering exons 2 and 3 of HLA-A, B and C, and exon 2 of DRB1, 3/4/5 and DQB1) and 96 MIDs in a single GS FLX run. An average of 166 reads per amplicon was obtained. We have also genotyped 96 samples at high resolution (14 primer pairs covering exons 2, 3, and 4 of the class I loci and exons 2 of DRB1, 3/4/5, DQA1, DQB1, DPB1, and exon 3 of DQB1), recovering an average of 173 sequence reads per amplicon.