Establishment of a Practical Workflow Using a Next Generation Sequencer to Search for Novel SNPs Associated with the Anti-HCV Treatment Response
Establishment of a Practical Workflow Using a Next Generation Sequencer to Search for Novel SNPs Associated with the Anti-HCV Treatment Response
复制标题
使用下一代测序仪建立实用工作流程来搜索与抗 HCV 治疗反应相关的新型 SNP
DOI：
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发表时间：
2014
期刊：
影响因子：
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通讯作者：
Sumio Watanabe
中科院分区：
文献类型：
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作者：
T. Takeda;M. Sugiyama;T. Kanto;M. Korenaga;M. Mizokami;Sumio Watanabe
目的：IL 28 B基因附近的单核苷酸多态性（SNPs）是慢性丙型肝炎患者对干扰素治疗反应的强预测因子。然而，大约20%的患者对治疗有不一致的反应，这表明未发现的SNP变异可能存在于该疾病相关基因附近。我们试图开发一种实用的方法，以探索基于下一代测序（NGS）技术的治疗反应中涉及的罕见变异。材质：8例IL 28 B SNP有利基因型（TT为rs 8099917）患者接受了48周的聚乙二醇干扰素-α和利巴韦林治疗，入组研究。根据其病毒学应答将其分为两组，5例达到早期病毒学应答（EVR），而其他患者则无病毒学应答（NVR）。方法：设计引物，特异性扩增IL 28 B及其相关基因IL 28 A。通过NGS对扩增子进行测序，并使用毛细管测序来验证通过NGS鉴定的变异。测量和结果：IL 28 B和IL 28 A周围的靶区域通过内部引物组特异性扩增。在NGS分析之前，引入实时PCR以控制每个样品上的序列读数的数目。通过EVR和NVR组的比较NGS分析，确定了四种候选罕见变异。为了验证NGS的结果，我们随后使用了毛细管测序，但未能证实这些罕见变异的存在。结论：我们已经用NGS建立了一个技术性的连续测序平台，有可能发现感兴趣的基因中的罕见变异SNP，例如HCV感染中的IL 28 B。
Objective: The single nucleotide polymorphisms (SNPs) close to the IL28B gene are strong predictors of response to interferon-based therapy for chronic hepatitis C patients. However, approximately 20% of patients have discordant responses to the therapy, suggesting that undiscovered variants of the SNPs may be present near to this disease-associated gene. We sought to develop a practical approach to explore rare variants involved in the treatment response, based on next-generation sequencing (NGS) technology. Materials: Eight patients with the favorable genotype of the IL28B SNP (TT of rs8099917) who underwent 48 week of pegylated interferon-α and ribavirin therapy were enrolled in the study. They were categorized in two groups according to their virological response, 5 achieved an early virological response (EVR) while the others had a null virological response (NVR). Methods: PCR primers were developed to amplify specifically IL28B and the relevant IL28A genes. The amplicons were sequenced by an NGS and capillary sequencing was used to validate the variations identified by NGS. Measurements and Results: Target regions around IL28B and IL28A were specifically amplified by the in house primer sets. Real-time PCR was introduced to control the number of sequence reads on each sample before NGS analysis. Four candidate rare variants were identified through comparative NGS analyses of the EVR and NVR groups. In order to validate the results of the NGS, we subsequently used capillary sequencing but failed to confirm the existence of these rare variants. Conclusions: We have established a technical serial sequencing platform with NGS, potentially enabling the discovery of rare variant SNPs in genes of interest, such as IL28B in HCV infection.
影响因子：
29.4
作者：
McHutchison, JG;Manns, M;Albrecht, JK
通讯作者：
Albrecht, JK