ILMN reads. MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome.

Advances in 3rd generation long read 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 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 tumour 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 Caenorhabditis elegans 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 flowcell and the best combined MinION data generated assembly produced a highly contiguous wild type C. elegans 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 C. elegans reference genome by >2Mb due to a more accurate determination of repetitive sequence elements, and assembled the complete genomes of two bacterial contaminants. MinION long read sequence data also facilitated the elucidation of complex rearrangements in the 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%.

第三代长读长测序技术的进步为台式全基因组测序开辟了全新可能。牛津纳米孔科技（Oxford Nanopore Technologies）MinION是一款采用纳米孔技术、可直接对DNA分子进行测序的便携式测序设备。MinION长读长序列非常适用于复杂基因组的从头组装（de novo assembly），因其可助力构建高度连续的物理基因组图谱，从而无需开展劳动密集型的物理基因组图谱绘制工作。长读长序列还可用于解析复杂的染色体重排事件——例如肿瘤细胞中出现的这类事件——这类事件往往会干扰短读长测序的分析结果。本研究针对MinION长读长序列的应用可行性展开评估，具体涵盖两大方向：1）大型复杂基因组的从头组装；2）复杂重排事件的解析。本研究采用MinION对两种秀丽隐杆线虫（Caenorhabditis elegans）品系的基因组进行测序：一种为野生型品系，另一种则携带两种复杂染色体重排。单条测序流通池即可获得最高42倍的测序覆盖度；基于最优MinION组合数据生成的组装结果，得到了高度连续的野生型秀丽隐杆线虫基因组：该组装包含48个重叠群（contig），其N50重叠群长度为3.99 Mb，覆盖了参考基因组（总长100,286,401个碱基）99%以上的序列。此外，由于更精准地解析了重复序列元件，本次MinION衍生的基因组组装结果较秀丽隐杆线虫参考基因组多出了超过2 Mb的序列；同时，本次组装还获得了两种污染细菌的完整基因组。MinION长读长序列数据同样助力解析了该诱变菌株中的复杂染色体重排事件。利用Illumina测序数据对最终基因组组装结果进行抛光处理，将MinION长读长重叠群的序列准确度（约98%）提升至99.8%。