Good Start Genetics, Cambridge, Massachusetts, USA.
Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
Genet Med. 2014 Feb;16(2):132-40. doi: 10.1038/gim.2013.83. Epub 2013 Jun 13.
Carrier screening for recessive Mendelian disorders traditionally employs focused genotyping to interrogate limited sets of mutations most prevalent in specific ethnic groups. We sought to develop a next-generation DNA sequencing-based workflow to enable analysis of a more comprehensive set of disease-causing mutations.
We utilized molecular inversion probes to capture the protein-coding regions of 15 genes from genomic DNA isolated from whole blood and sequenced those regions using the Illumina HiSeq 2000 (Illumina, San Diego, CA). To assess the quality of the resulting data, we measured both the fraction of the targeted region yielding high-quality genotype calls, and the sensitivity and specificity of those calls by comparison with conventional Sanger sequencing across hundreds of samples. Finally, to improve the overall accuracy for detecting insertions and deletions, we introduce a novel assembly-based approach that substantially increases sensitivity without reducing specificity.
We generated high-quality sequence for at least 99.8% of targeted base pairs in samples derived from blood and achieved high concordance with Sanger sequencing (sensitivity >99.9%, specificity >99.999%). Our novel algorithm is capable of detecting insertions and deletions inaccessible by current methods.
Our next-generation DNA sequencing-based approach yields the accuracy and completeness necessary for a carrier screening test.
传统的隐性孟德尔疾病携带者筛查采用靶向基因分型技术，针对特定族群中最常见的有限突变进行检测。我们试图开发一种基于新一代 DNA 测序的工作流程，以实现对更广泛的致病突变的分析。
我们使用分子反转探针从全血中提取基因组 DNA 的蛋白质编码区域，并使用 Illumina HiSeq 2000（Illumina，圣地亚哥，CA）对这些区域进行测序。为了评估所得数据的质量，我们通过与数百个样本的传统 Sanger 测序进行比较，测量了目标区域产生高质量基因型的比例，以及这些基因型的灵敏度和特异性。最后，为了提高检测插入和缺失的整体准确性，我们引入了一种新颖的基于组装的方法，该方法在不降低特异性的情况下显著提高了灵敏度。
我们从血液样本中生成了至少 99.8%靶向碱基对的高质量序列，并且与 Sanger 测序高度一致（灵敏度>99.9%，特异性>99.999%）。我们的新算法能够检测到当前方法无法检测到的插入和缺失。
我们基于新一代 DNA 测序的方法能够提供进行携带者筛查测试所需的准确性和完整性。
