Next generation sequencing (NGS) has changed our approach to diagnosis of genetic disorders. Nowadays, the most comprehensive application of NGS is whole genome sequencing (WGS) that is able to detect virtually all DNA variations. However, even after accurate WGS, many genetic conditions remain unsolved. This may be due to the current NGS protocols, based on DNA fragmentation and short reads. To overcome these limitations, we applied a linked-read sequencing technology that combines single-molecule barcoding with short-read WGS. We were able to assemble haplotypes and distinguish between alleles along the genome. As an exemplary case, we studied the case of a female carrier of X-linked muscular dystrophy with an unsolved genetic status. A deletion of exons 16-29 in DMD gene was responsible for the disease in her family, but she showed a normal dosage of these exons by Multiplex Ligation-dependent Probe Amplification (MLPA) and array CGH. This situation is usually considered compatible with a "non-carrier" status. Unexpectedly, the girl also showed an increased dosage of flanking exons 1-15 and 30-34. Using linked-read WGS, we were able to distinguish between the two X chromosomes. In the first allele, we found the 16-29 deletion, while the second allele showed a 1-34 duplication: in both cases, linked-read WGS correctly mapped the borders at single-nucleotide resolution. This duplication in trans apparently restored the normal dosage of exons 16-29 seen by quantitative assays. This had a dramatic impact in genetic counselling, by converting a non-carrier into a double carrier status prediction. We conclude that linked-read WGS should be considered as a valuable option to improve our understanding of unsolved genetic conditions.

中文翻译：

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Linked-Read 全基因组测序解决了双重 DMD 基因重排

下一代测序 (NGS) 改变了我们诊断遗传疾病的方法。如今，NGS 最全面的应用是全基因组测序 (WGS)，它能够检测几乎所有的 DNA 变异。然而，即使在准确的 WGS 之后，许多遗传条件仍未解决。这可能是由于当前基于 DNA 片段化和短读长的 NGS 协议所致。为了克服这些限制，我们应用了一种链接读取测序技术，该技术将单分子条形码与短读 WGS 相结合。我们能够组装单倍型并区分基因组中的等位基因。作为一个典型案例，我们研究了一名 X 连锁肌营养不良症女性携带者的案例，其遗传状态尚未解决。DMD 基因中外显子 16-29 的缺失是导致她家族患病的原因，但她通过多重连接依赖探针扩增 (MLPA) 和阵列 CGH 显示出这些外显子的正常剂量。这种情况通常被认为与“非承运人”状态兼容。出乎意料的是，女孩还表现出侧翼外显子1-15和30-34的剂量增加。使用链接读取 WGS，我们能够区分两条 X 染色体。在第一个等位基因中，我们发现了 16-29 缺失，而第二个等位基因显示了 1-34 重复：在这两种情况下，链接读取 WGS 都以单核苷酸分辨率正确绘制了边界。这种反式重复显然恢复了定量分析所见的外显子 16-29 的正常剂量。通过将非携带者转换为双重携带者状态预测，这对遗传咨询产生了巨大影响。