Department of Neuroscience, Mayo Clinic, Jacksonville, FL, 32224, USA.
Mayo Graduate School, Mayo Clinic, Rochester, MN, 55905, USA.
Mol Neurodegener. 2018 Aug 21;13(1):46. doi: 10.1186/s13024-018-0274-4.
Many neurodegenerative diseases are caused by nucleotide repeat expansions, but most expansions, like the C9orf72 'GGGGCC' (GC) repeat that causes approximately 5-7% of all amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases, are too long to sequence using short-read sequencing technologies. It is unclear whether long-read sequencing technologies can traverse these long, challenging repeat expansions. Here, we demonstrate that two long-read sequencing technologies, Pacific Biosciences' (PacBio) and Oxford Nanopore Technologies' (ONT), can sequence through disease-causing repeats cloned into plasmids, including the FTD/ALS-causing GC repeat expansion. We also report the first long-read sequencing data characterizing the C9orf72 GC repeat expansion at the nucleotide level in two symptomatic expansion carriers using PacBio whole-genome sequencing and a no-amplification (No-Amp) targeted approach based on CRISPR/Cas9.
Both the PacBio and ONT platforms successfully sequenced through the repeat expansions in plasmids. Throughput on the MinION was a challenge for whole-genome sequencing; we were unable to attain reads covering the human C9orf72 repeat expansion using 15 flow cells. We obtained 8× coverage across the C9orf72 locus using the PacBio Sequel, accurately reporting the unexpanded allele at eight repeats, and reading through the entire expansion with 1324 repeats (7941 nucleotides). Using the No-Amp targeted approach, we attained > 800× coverage and were able to identify the unexpanded allele, closely estimate expansion size, and assess nucleotide content in a single experiment. We estimate the individual's repeat region was > 99% GC content, though we cannot rule out small interruptions.
Our findings indicate that long-read sequencing is well suited to characterizing known repeat expansions, and for discovering new disease-causing, disease-modifying, or risk-modifying repeat expansions that have gone undetected with conventional short-read sequencing. The PacBio No-Amp targeted approach may have future potential in clinical and genetic counseling environments. Larger and deeper long-read sequencing studies in C9orf72 expansion carriers will be important to determine heterogeneity and whether the repeats are interrupted by non-GC content, potentially mitigating or modifying disease course or age of onset, as interruptions are known to do in other repeat-expansion disorders. These results have broad implications across all diseases where the genetic etiology remains unclear.
许多神经退行性疾病是由核苷酸重复扩展引起的，但大多数扩展，如导致大约 5-7%的所有肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）病例的 C9orf72“GGGGCC”（GC）重复，都太长了，无法使用短读测序技术进行测序。目前尚不清楚长读测序技术是否可以跨越这些长而具有挑战性的重复扩展。在这里，我们证明了两种长读测序技术，即 Pacific Biosciences（PacBio）和 Oxford Nanopore Technologies（ONT），可以对克隆到质粒中的致病重复进行测序，包括导致 FTD/ALS 的 GC 重复扩展。我们还报告了首次使用 PacBio 全基因组测序和基于 CRISPR/Cas9 的无扩增（No-Amp）靶向方法在两个有症状的扩展携带者中对 C9orf72 GC 重复进行核苷酸水平特征描述的长读测序数据。
PacBio 和 ONT 平台都成功地对质粒中的重复扩展进行了测序。MinION 的通量对全基因组测序来说是一个挑战；我们无法使用 15 个流池获得覆盖人类 C9orf72 重复扩展的读数。我们使用 PacBio Sequel 获得了 C9orf72 基因座的 8×覆盖，准确地报告了 8 个重复的未扩展等位基因，并通过 1324 个重复（7941 个核苷酸）读取整个扩展。使用 No-Amp 靶向方法，我们获得了 >800×的覆盖，并能够在单个实验中识别未扩展的等位基因，准确估计扩展大小，并评估核苷酸含量。我们估计个体的重复区域 >99%的 GC 含量，尽管我们不能排除小的中断。
我们的发现表明，长读测序非常适合于描述已知的重复扩展，并且可用于发现新的致病、疾病修饰或风险修饰的重复扩展，这些扩展在常规短读测序中未被发现。PacBio No-Amp 靶向方法在临床和遗传咨询环境中可能具有未来的潜力。在 C9orf72 扩展携带者中进行更大和更深的长读测序研究将很重要，以确定异质性以及重复是否被非 GC 含量中断，这可能减轻或修饰疾病过程或发病年龄，因为中断在其他重复扩展疾病中是已知的。这些结果对所有遗传病因仍不清楚的疾病都具有广泛的意义。
