Nanopore 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的应用前景。牛津纳米孔科技公司（Oxford Nanopore Technologies）推出的MinION测序仪是一款依托纳米孔技术的便携式设备，可直接对DNA分子完成测序。MinION长读长序列十分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，省去了耗时耗力的物理基因组图谱构建流程。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重排，这类事件往往会对短读长测序的分析结果造成干扰。本研究针对MinION长读长序列的应用可行性展开了评估，涵盖两大方向：1）大型复杂基因组的从头组装；2）复杂染色体重排的解析。研究人员使用MinION对两种秀丽隐杆线虫（Caenorhabditis elegans）菌株的基因组进行了测序，分别为野生型菌株与携带两种复杂染色体重排的诱变菌株。单次测序流槽（flowcell）即可获得最高达42倍的测序覆盖度；最优的MinION联合数据组装结果得到了高度连续的野生型秀丽隐杆线虫基因组，共包含48个重叠群（contig），重叠群N50长度达3.99 Mb，覆盖了长度为100,286,401个碱基的参考基因组超过99%的序列。此外，得益于对重复序列元件的精准测定，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，同时还组装出了两种污染菌的完整基因组。MinION长读长序列数据还助力解析了该诱变菌株中的复杂染色体重排事件。研究人员借助Illumina测序数据对MinION长读长重叠群进行序列抛光，将其序列准确度（约98%）提升至99.8%，最终得到了优化后的基因组组装结果。