Institute of Clinical Chemistry, Inselspital, University Hospital and University of Bern, Bern, Switzerland.
Clin Chem. 2011 Jan;57(1):102-11. doi: 10.1373/clinchem.2010.150706. Epub 2010 Nov 10.
Molecular genetic testing is commonly used to confirm clinical diagnoses of inherited urea cycle disorders (UCDs); however, conventional mutation screenings encompassing only the coding regions of genes may not detect disease-causing mutations occurring in regulatory elements and introns. Microarray-based target enrichment and next-generation sequencing now allow more-comprehensive genetic screening. We applied this approach to UCDs and combined it with the use of DNA bar codes for more cost-effective, parallel analyses of multiple samples.
We used sectored 2240-feature medium-density oligonucleotide arrays to capture and enrich a 199-kb genomic target encompassing the complete genomic regions of 3 urea cycle genes, OTC (ornithine carbamoyltransferase), CPS1 (carbamoyl-phosphate synthetase 1, mitochondrial), and NAGS (N-acetylglutamate synthase). We used the Genome Sequencer FLX System (454 Life Sciences) to jointly analyze 4 samples individually tagged with a 6-bp DNA bar code and compared the results with those for an individually sequenced sample.
Using a low tiling density of only 1 probe per 91 bp, we obtained strong enrichment of the targeted loci to achieve ≥90% coverage with up to 64% of the sequences covered at a sequencing depth ≥10-fold. We observed a very homogeneous sequence representation of the bar-coded samples, which yielded a >30% increase in the sequence data generated per sample, compared with an individually processed sample. Heterozygous and homozygous disease-associated mutations were correctly detected in all samples.
The use of DNA bar codes and the use of sectored oligonucleotide arrays for target enrichment enable parallel, large-scale analysis of complete genomic regions for multiple genes of a disease pathway and for multiple samples simultaneously. This approach thus may provide an efficient tool for comprehensive diagnostic screening of mutations.
分子遗传学检测常用于确认遗传性尿素循环障碍（UCD）的临床诊断；然而，仅涵盖基因编码区的传统突变筛查可能无法检测到发生在调控元件和内含子中的致病突变。基于微阵列的靶向富集和新一代测序现在可以实现更全面的基因筛查。我们将这种方法应用于 UCD，并结合使用 DNA 条码，以更具成本效益的方式对多个样本进行并行分析。
我们使用扇形 2240 个特征中密度寡核苷酸阵列来捕获和富集一个 199-kb 的基因组靶标，该靶标包含 3 个尿素循环基因（OTC[鸟氨酸氨甲酰基转移酶]、CPS1[氨甲酰磷酸合成酶 1，线粒体]和 NAGS[N-乙酰谷氨酸合酶]）的完整基因组区域。我们使用 Genome Sequencer FLX System（454 Life Sciences）联合分析 4 个单独标记有 6 个碱基对 DNA 条码的样本，并将结果与单独测序样本的结果进行比较。
使用仅每 91bp 有 1 个探针的低覆盖度，我们实现了靶向区域的强富集，以实现≥90%的覆盖度，多达 64%的序列在测序深度≥10 倍时被覆盖。我们观察到条码样本的序列表示非常均匀，与单独处理的样本相比，每个样本生成的序列数据增加了>30%。所有样本均正确检测到杂合和纯合的疾病相关突变。
使用 DNA 条码和扇形寡核苷酸阵列进行靶向富集可以同时对疾病途径的多个基因和多个样本进行并行、大规模的全基因组区域分析。这种方法因此可能为突变的综合诊断筛查提供有效的工具。
