Biomolecular Screening Branch, National Institute of Environmental Health Sciences, 111 T.W. Alexander Dr. Research Triangle Park, Durham, NC, USA.
Sciome, LLC, Research Triangle Park, Durham, NC, USA.
BMC Genomics. 2018 Jun 20;19(1):487. doi: 10.1186/s12864-018-4858-8.
The rat genome was sequenced in 2004 with the aim to improve human health altered by disease and environmental influences through gene discovery and animal model validation. Here, we report development and testing of a probe set for whole exome sequencing (WES) to detect sequence variants in exons and UTRs of the rat genome. Using an in-silico approach, we designed probes targeting the rat exome and compared captured mutations in cancer-related genes from four chemically induced rat tumor cell lines (C6, FAT7, DSL-6A/C1, NBTII) to validated cancer genes in the human database, Catalogue of Somatic Mutations in Cancer (COSMIC) as well as normal rat DNA. Paired, fresh frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) liver tissue from naive rats were sequenced to confirm known dbSNP variants and identify any additional variants.
Informatics analysis of available gene annotation from rat RGSC6.0/rn6 RefSeq and Ensembl transcripts provided 223,636 unique exons representing a total of 26,365 unique genes and untranslated regions. Using this annotation and the Rn6 reference genome, an in-silico probe design generated 826,878 probe sequences of which 94.2% were uniquely aligned to the rat genome without mismatches. Further informatics analysis revealed 25,249 genes (95.8%) covered by at least one probe and 23,603 genes (93.5%) had every exon covered by one or more probes. We report high performance metrics from exome sequencing of our probe set and Sanger validation of annotated, highly relevant, cancer gene mutations as cataloged in the human COSMIC database, in addition to several exonic variants in cancer-related genes.
An in-silico probe set was designed to enrich the rat exome from isolated DNA. The platform was tested on rat tumor cell lines and normal FF and FFPE liver tissue. The method effectively captured target exome regions in the test DNA samples with exceptional sensitivity and specificity to obtain reliable sequencing data representing variants that are likely chemically induced somatic mutations. Genomic discovery conducted by means of high throughput WES queries should benefit investigators in discovering rat genomic variants in disease etiology and in furthering human translational research.
2004 年，老鼠基因组测序的目的是通过基因发现和动物模型验证，改善受疾病和环境影响的人类健康。在这里，我们报告了一种用于外显子组测序（WES）的探针集的开发和测试，以检测大鼠基因组中外显子和 UTR 中的序列变异。我们使用一种计算机方法，设计了针对大鼠外显子的探针，并比较了来自四种化学诱导的大鼠肿瘤细胞系（C6、FAT7、DSL-6A/C1、NBTII）的癌症相关基因中捕获的突变与人类数据库中的癌症基因、癌症体细胞突变目录（COSMIC）以及正常大鼠 DNA。对来自天真大鼠的配对新鲜冷冻（FF）和福尔马林固定、石蜡包埋（FFPE）肝脏组织进行测序，以确认已知的 dbSNP 变体并确定任何其他变体。
对来自大鼠 RGSC6.0/rn6 RefSeq 和 Ensembl 转录本的现有基因注释的信息学分析提供了 223636 个独特的外显子，代表了总共 26365 个独特的基因和非翻译区。使用此注释和 rn6 参考基因组，计算机探针设计生成了 826878 个探针序列，其中 94.2%与大鼠基因组无错配地唯一对齐。进一步的信息学分析显示，至少有一个探针覆盖了 25249 个基因（95.8%），并且 23603 个基因（93.5%）的每个外显子都由一个或多个探针覆盖。我们报告了我们的探针集在外显子组测序中的高性能指标，并对人类 COSMIC 数据库中编目的高度相关的癌症基因突变更正，以及癌症相关基因中的几个外显子变体进行了 Sanger 验证。
设计了一种计算机探针集，用于从分离的 DNA 中富集大鼠外显子。该平台在大鼠肿瘤细胞系和正常 FF 和 FFPE 肝脏组织上进行了测试。该方法有效地捕获了测试 DNA 样本中的目标外显子区域，具有出色的灵敏度和特异性，可获得可靠的测序数据，代表可能是化学诱导的体细胞突变的变体。通过高通量外显子组测序进行的基因组发现应该使研究人员受益于发现疾病病因中的大鼠基因组变体，并推进人类转化研究。
