Design and implementation of a custom next generation sequencing panel for selected vitamin D associated genes.
Design and implementation of a custom next generation sequencing panel for selected vitamin D associated genes.
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DOI：
10.1186/s13104-017-2664-z
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发表时间：
2017-07-28
影响因子：
1.8
通讯作者：
McKnight AJ
中科院分区：
文献类型：
--
作者：
Benson KA;Chand S;Maxwell AP;Smyth LJ;Kilner J;Borrows R;McKnight AJ
具有生物活性的维生素D在基因组中具有重要的调节作用。它结合维生素D受体（VDR）以控制多种基因的表达，并与表观基因组相互作用以改变染色质和甲基化状态。维生素D缺乏与包括终末期肾病在内的几种人类疾病有关。本文描述了定制的设计和测试，针对选定的维生素D相关基因的下一代测序（NGS）面板。测序运行用于确定变异调用面板的有效性，比较不同测序器的效率和数据，并执行具有代表性的原则关联分析。这些分析不足以进行显著性检验。扩增子分别在两个池（163和166个片段）中设计，并在Ion Personal Genome Machine （PGM）™和Ion S5™XL台式测序仪上对两组肾移植受者进行测序。覆盖了7个维生素D相关基因（CYP24A1， CUBN， VDR， GC, NADSYN1, CYP27B1, CYP2R1）的43.8千碱基以及38个优先snp。测序运行提供了足够的测序质量，数据输出，并验证了有效的文库制备和面板设计。这种新颖的，定制设计的，经过验证的面板为广泛的患者队列中维生素D相关基因的分析提供了一种快速，具有成本效益和特异性的方法。本文未报道受控卫生保健干预的结果。本文的在线版本（doi:10.1186/s13104-017-2664-z）包含补充材料，可供授权用户使用。
Biologically active vitamin D has an important regulatory role within the genome. It binds the vitamin D receptor (VDR) in order to control the expression of a wide range of genes as well as interacting with the epigenome to modify chromatin and methylation status. Vitamin D deficiency is associated with several human diseases including end-stage renal disease. This article describes the design and testing of a custom, targeted next generation sequencing (NGS) panel for selected vitamin D associated genes. Sequencing runs were used to determine the effectiveness of the panel for variant calling, to compare efficiency and data across different sequencers, and to perform representative, proof of principle association analyses. These analyses were underpowered for significance testing. Amplicons were designed in two pools (163 and 166 fragments respectively) and used to sequence two cohorts of renal transplant recipients on the Ion Personal Genome Machine (PGM)™ and Ion S5™ XL desktop sequencers. Coverage was provided for 43.8 kilobases across seven vitamin D associated genes (CYP24A1, CUBN, VDR, GC, NADSYN1, CYP27B1, CYP2R1) as well as 38 prioritised SNPs. Sequencing runs provided sufficient sequencing quality, data output and validated the effective library preparation and panel design. This novel, custom-designed, validated panel provides a fast, cost effective, and specific approach for the analysis of vitamin D associated genes in a wide range of patient cohorts. This article does not report results from a controlled health-care intervention. The online version of this article (doi:10.1186/s13104-017-2664-z) contains supplementary material, which is available to authorized users.