Michael Smith Laboratories and Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4.
Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4.
Genome Res. 2018 Feb;28(2):266-274. doi: 10.1101/gr.221184.117. Epub 2017 Dec 22.
Advances in long-read single molecule sequencing have opened new possibilities for 'benchtop' whole-genome sequencing. The Oxford Nanopore Technologies MinION is a portable device that uses nanopore technology that can directly sequence DNA molecules. MinION single molecule long sequence reads are well suited for de novo assembly of complex genomes as they facilitate the construction of highly contiguous physical genome maps obviating the need for labor-intensive physical genome mapping. Long sequence reads can also be used to delineate complex chromosomal rearrangements, such as those that occur in tumor cells, that can confound analysis using short reads. Here, we assessed MinION long-read-derived sequences for feasibility concerning: (1) the de novo assembly of a large complex genome, and (2) the elucidation of complex rearrangements. The genomes of two strains, a wild-type strain and a strain containing two complex rearrangements, were sequenced with MinION. Up to 42-fold coverage was obtained from a single flow cell, and the best pooled data assembly produced a highly contiguous wild-type genome containing 48 contigs (N50 contig length = 3.99 Mb) covering >99% of the 100,286,401-base reference genome. Further, the MinION-derived genome assembly expanded the reference genome by >2 Mb due to a more accurate determination of repetitive sequence elements and assembled the complete genomes of two co-extracted bacteria. MinION long-read sequence data also facilitated the elucidation of complex rearrangements in a mutagenized strain. The sequence accuracy of the MinION long-read contigs (∼98%) was improved using Illumina-derived sequence data to polish the final genome assembly to 99.8% nucleotide accuracy when compared to the reference assembly.
长读单分子测序技术的进步为“台式”全基因组测序开辟了新的可能性。Oxford Nanopore Technologies MinION 是一种便携式设备，它使用纳米孔技术，可以直接对 DNA 分子进行测序。MinION 单分子长序列读取非常适合从头组装复杂基因组，因为它们有助于构建高度连续的物理基因组图谱，从而避免了劳动密集型的物理基因组图谱构建。长序列读取还可用于描绘复杂的染色体重排，例如发生在肿瘤细胞中的那些重排，这些重排可能会干扰使用短读长进行的分析。在这里，我们评估了 MinION 长读序列在以下方面的可行性：(1) 大型复杂基因组的从头组装，和 (2) 复杂重排的阐明。用 MinION 对两种 菌株的基因组进行了测序，一种是野生型菌株，另一种是含有两种复杂重排的菌株。从单个流动池中获得了高达 42 倍的覆盖率，最佳的合并数据组装生成了一个高度连续的野生型 基因组，包含 48 个 contigs（N50 contig 长度=3.99 Mb），覆盖了 100,286,401 个碱基参考基因组的>99%。此外，由于更准确地确定了重复序列元件，MinION 衍生的基因组组装使 参考基因组扩展了>2 Mb，并组装了两个共提取细菌的完整基因组。MinION 长读序列数据还有助于阐明诱变菌株中的复杂重排。使用 Illumina 衍生的序列数据对 MinION 长读 contigs 的序列准确性（约 98%）进行了改进，与参考组装相比，将最终基因组组装的核苷酸准确性提高到 99.8%。
