Efficient In Silico Identification of a Common Insertion in the MAK Gene which Causes Retinitis Pigmentosa.
Efficient In Silico Identification of a Common Insertion in the MAK Gene which Causes Retinitis Pigmentosa.
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DOI：
10.1371/journal.pone.0142614
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Comander J
中科院分区：
文献类型：
--
作者：
Bujakowska KM;White J;Place E;Consugar M;Comander J
下一代测序 (NGS) 提供了一种快速、全面的方法来筛查与色素性视网膜炎和相关疾病相关的突变。然而，使用标准 NGS 流程可能无法检测到某些序列改变，例如大插入或缺失。其中一种突变是最近发现的雄性生殖细胞相关激酶 (MAK) 基因中的 Alu 插入，这是基于标准 NGS 的变异识别器所遗漏的。在这里，我们开发了一种计算机方法，可以搜索 NGS 原始序列读数来检测这种突变，而无需重新计算序列比对或通过 PCR 筛选每个样本。 Linux 程序 grep 用于搜索包含已知插入片段连接序列的 23 bp“探针”序列。使用野生型序列进行相应的搜索。对匹配的读数进行计数，并进一步与完整野生型和突变基因组基因座的已知序列进行比较。 （参见 https://github.com/MEEIBioinformaticsCenter/grepsearch。）在由 11 个先前发表的纯合突变体组成的测试样本集中，MAK-Alu 插入的检测经过验证，具有 100% 的灵敏度和特异性。作为一个发现队列，在本地计算机集群上约 1 小时内搜索了 1,847 个样本（包括定制和全外显子组选择性捕获）的原始 NGS 读数，产生了另外 5 个带有 MAK-Alu 插入的样本，并解决了两个以前未解决的谱系问题。其中，一名患者为插入纯合子，一名患者为在另一个等位基因上存在错义变化的复合杂合子（c.46G>A；p.Gly16Arg），三名患者为杂合子携带者。事实证明，使用 MAK-Alu grep 程序是一种快速有效的方法，可以在大量具有 NGS 数据的患者中找到已知的致病 Alu 插入。这种简单的方法避免了湿实验室测定或计算成本昂贵的算法，并且也可用于其他已知的致病插入和缺失。
Next generation sequencing (NGS) offers a rapid and comprehensive method of screening for mutations associated with retinitis pigmentosa and related disorders. However, certain sequence alterations such as large insertions or deletions may remain undetected using standard NGS pipelines. One such mutation is a recently-identified Alu insertion into the Male Germ Cell-Associated Kinase (MAK) gene, which is missed by standard NGS-based variant callers. Here, we developed an in silico method of searching NGS raw sequence reads to detect this mutation, without the need to recalculate sequence alignments or to screen every sample by PCR. The Linux program grep was used to search for a 23 bp “probe” sequence containing the known junction sequence of the insert. A corresponding search was performed with the wildtype sequence. The matching reads were counted and further compared to the known sequences of the full wildtype and mutant genomic loci. (See https://github.com/MEEIBioinformaticsCenter/grepsearch.) In a test sample set consisting of eleven previously published homozygous mutants, detection of the MAK-Alu insertion was validated with 100% sensitivity and specificity. As a discovery cohort, raw NGS reads from 1,847 samples (including custom and whole exome selective capture) were searched in ~1 hour on a local computer cluster, yielding an additional five samples with MAK-Alu insertions and solving two previously unsolved pedigrees. Of these, one patient was homozygous for the insertion, one compound heterozygous with a missense change on the other allele (c. 46G>A; p.Gly16Arg), and three were heterozygous carriers. Using the MAK-Alu grep program proved to be a rapid and effective method of finding a known, disease-causing Alu insertion in a large cohort of patients with NGS data. This simple approach avoids wet-lab assays or computationally expensive algorithms, and could also be used for other known disease-causing insertions and deletions.