Liem Michael, Jansen Hans J, Dirks Ron P, Henkel Christiaan V, van Heusden G Paul H, Lemmers Richard J L F, Omer Trifa, Shao Shuai, Punt Peter J, Spaink Herman P
Institute of Biology, Leiden University, Leiden, 2300 RA, Netherlands.
Future Genomics Technologies B.V., Leiden, 2333 BE, Netherlands.
F1000Res. 2017 May 3;6:618. doi: 10.12688/f1000research.11146.2. eCollection 2017.
The introduction of the MinION sequencing device by Oxford Nanopore Technologies may greatly accelerate whole genome sequencing. Nanopore sequence data offers great potential for assembly of complex genomes without using other technologies. Furthermore, Nanopore data combined with other sequencing technologies is highly useful for accurate annotation of all genes in the genome. In this manuscript we used nanopore sequencing as a tool to classify yeast strains. We compared various technical and software developments for the nanopore sequencing protocol, showing that the R9 chemistry is, as predicted, higher in quality than R7.3 chemistry. The R9 chemistry is an essential improvement for assembly of the extremely AT-rich mitochondrial genome. We double corrected assemblies from four different assemblers with PILON and assessed sequence correctness before and after PILON correction with a set of 290 Fungi genes using BUSCO. In this study, we used this new technology to sequence and assemble the genome of a recently isolated ethanologenic yeast strain, and compared the results with those obtained by classical Illumina short read sequencing. This strain was originally named ( ) based on ribosomal RNA sequencing. We show that the assembly using nanopore data is much more contiguous than the assembly using short read data. We also compared various technical and software developments for the nanopore sequencing protocol, showing that nanopore-derived assemblies provide the highest contiguity. The mitochondrial and chromosomal genome sequences showed that our strain is clearly distinct from other yeast taxons and most closely related to published species. In conclusion, MinION-mediated long read sequencing can be used for high quality assembly of new eukaryotic microbial genomes.
牛津纳米孔技术公司推出的MinION测序设备可能会极大地加速全基因组测序。纳米孔序列数据为不使用其他技术组装复杂基因组提供了巨大潜力。此外，纳米孔数据与其他测序技术相结合对于基因组中所有基因的准确注释非常有用。在本论文中，我们使用纳米孔测序作为工具对酵母菌株进行分类。我们比较了纳米孔测序方案的各种技术和软件开发，结果表明，正如预期的那样，R9化学技术的质量高于R7.3化学技术。R9化学技术对于组装富含AT的线粒体基因组来说是一项至关重要的改进。我们使用PILON对来自四种不同组装器的组装结果进行了双重校正，并使用一组290个真菌基因通过BUSCO评估了PILON校正前后的序列正确性。在本研究中，我们使用这项新技术对最近分离的产乙醇酵母菌株的基因组进行测序和组装，并将结果与通过经典的Illumina短读长测序获得的结果进行比较。该菌株最初根据核糖体RNA测序被命名为（ ）。我们表明，使用纳米孔数据进行的组装比使用短读长数据进行的组装更具连续性。我们还比较了纳米孔测序方案的各种技术和软件开发，结果表明纳米孔衍生的组装提供了最高的连续性。线粒体和染色体基因组序列表明，我们的菌株与其他酵母分类群明显不同，并且与已发表的物种关系最为密切。总之，MinION介导的长读长测序可用于新的真核微生物基因组的高质量组装。
