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首页> 外文期刊>International Journal of Molecular Epidemiology and Genetics >Next generation sequencing of CLU, PICALM and CR1: pitfalls and potential solutions
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Next generation sequencing of CLU, PICALM and CR1: pitfalls and potential solutions

机译：CLU，PICALM和CR1的下一代测序：陷阱和潜在解决方案

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emCLU/em, emPICALM/em and emCR1/em were identified as genetic risk factors for late onset Alzheimer’s disease (AD) in two large genome wide association studies (GWAS) published in 2009, but the variants that convey this alteration in disease risk, and how the genes relate to AD pathology is yet to be discovered. A next generation sequencing (NGS) project was conducted targeting emCLU/em, emCR1/em and emPICALM/em, in 96 AD samples (8 pools of 12), in an attempt to discover rare variants within these AD associated genes. Inclusion of repetitive regions in the design of the SureSelect capture lead to significant issues in alignment of the data, leading to poor specificity and a lower than expected depth of coverage. A strong positive correlation (0.964, p<0.001) was seen between NGS and 1000 genome project frequency estimates. Of the ~170 “novel” variants detected in the genes, seven SNPs, all of which were present in multiple sample pools, were selected for validation by Sanger sequencing. Two SNPs were successfully validated by this method, and shown to be genuine variants, while five failed validation. These spurious SNP calls occurred as a result of the presence of small indels and mononucleotide repeats, indicating such features should be regarded with caution, and validation via an independent method is important for NGS variant calls.
机译：在两个大型的全基因组关联中，将 CLU ， PICALM 和 CR1 确定为迟发性阿尔茨海默氏病（AD）的遗传危险因素研究（GWAS）于2009年发表，但仍未发现能够传达这种疾病风险改变以及基因与AD病理学关系如何的变体。在96个AD样本（8个库，共12个）中，针对 CLU ， CR1 和 PICALM 进行了下一代测序（NGS）项目。试图在这些AD相关基因中发现稀有变体。 SureSelect捕获的设计中包含重复区域会导致数据对齐方面出现重大问题，从而导致特异性差且覆盖深度低于预期范围。在NGS和1000个基因组计划频率估算值之间发现了很强的正相关（0.964，p＆＃x0003c; 0.001）。 〜170＆＃x0201c; novel＆＃x0201d;通过Sanger测序，选择了在基因中检测到的7个SNP（都存在于多个样品池中）的变异体进行验证。通过这种方法成功验证了两个SNP，并显示是真正的变异，而五个验证失败。这些杂散的SNP调用是由于存在小插入缺失和单核苷酸重复而导致的，表​​明应谨慎考虑此类特征，并且通过独立方法进行的验证对于NGS变异调用非常重要。

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