Nanopore sequencing detects structural variants in cancer.
Nanopore sequencing detects structural variants in cancer.
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DOI：
10.1080/15384047.2016.1139236
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发表时间：
2016
影响因子：
3.6
通讯作者：
Timp W
中科院分区：
文献类型：
--
作者：
Norris AL;Workman RE;Fan Y;Eshleman JR;Timp W
尽管测序技术取得了进步，但由于第二代测序的读长较短（<300 bp），结构变异 (SV) 仍然难以可靠检测。不仅读数（或双端读数）需要跨越断点，而且重复元素通常会导致短读数的对齐不明确。我们建议使用第三代测序可能的长读长（高达 20 kb），特别是 MinION 上的纳米孔测序。纳米孔测序依赖于与库尔特计数器类似的概念，通过 DNA 链被迫穿过嵌入膜中的纳米大小的孔而产生的电流变化来读取 DNA 序列。虽然纳米孔测序目前错配率较高，无法检测碱基替换和小移码突变，但由于读长较长，其准确性足以用于SV检测。事实上，某些情况下长读可能会提高 SV 检测效率。我们测试了纳米孔测序，以检测一系列特征明确的 SV，包括导致胰腺癌中 CDKN2A/p16 和 SMAD4/DPC4 肿瘤抑制基因失活的大缺失、倒位和易位。使用 PCR 扩增子混合物，我们已经证明纳米孔测序可以在低至 1:100 的稀释度下检测大的缺失、易位和倒位，每个样本的读数少至 500 个。鉴于速度快、占地面积小和资本成本低，纳米孔测序可能成为低水平检测分子复发、早期检测或治疗监测所需的癌症相关 SV 的理想工具。
Despite advances in sequencing, structural variants (SVs) remain difficult to reliably detect due to the short read length (<300 bp) of 2nd generation sequencing. Not only do the reads (or paired-end reads) need to straddle a breakpoint, but repetitive elements often lead to ambiguities in the alignment of short reads. We propose to use the long-reads (up to 20 kb) possible with 3rd generation sequencing, specifically nanopore sequencing on the MinION. Nanopore sequencing relies on a similar concept to a Coulter counter, reading the DNA sequence from the change in electrical current resulting from a DNA strand being forced through a nanometer-sized pore embedded in a membrane. Though nanopore sequencing currently has a relatively high mismatch rate that precludes base substitution and small frameshift mutation detection, its accuracy is sufficient for SV detection because of its long reads. In fact, long reads in some cases may improve SV detection efficiency. We have tested nanopore sequencing to detect a series of well-characterized SVs, including large deletions, inversions, and translocations that inactivate the CDKN2A/p16 and SMAD4/DPC4 tumor suppressor genes in pancreatic cancer. Using PCR amplicon mixes, we have demonstrated that nanopore sequencing can detect large deletions, translocations and inversions at dilutions as low as 1:100, with as few as 500 reads per sample. Given the speed, small footprint, and low capital cost, nanopore sequencing could become the ideal tool for the low-level detection of cancer-associated SVs needed for molecular relapse, early detection, or therapeutic monitoring.