Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany; The Bioeconomy Science Center (BioSC), c/o Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.
IBMG: Institute for Biology I, RWTH Aachen University, Worringer Weg 2, 52074 Aachen, Germany; IBG-2 Plant Sciences, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany; The Bioeconomy Science Center (BioSC), c/o Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.
J Biotechnol. 2017 Sep 20;258:197-205. doi: 10.1016/j.jbiotec.2017.04.016. Epub 2017 Apr 19.
State of the art and novel high-throughput DNA sequencing technologies enable fascinating opportunities and applications in the life sciences including microbial genomics. Short high-quality read data already enable not only microbial genome sequencing, yet can be inadequately to solve problems in genome assemblies and for the analysis of structural variants, especially in engineered microbial cell factories. Single-molecule real-time sequencing technologies generating long reads promise to solve such assembly problems. In our study, we wanted to increase the average read length of long nanopore reads with R9 chemistry and conducted a hybrid approach for the analysis of structural variants to check the genome stability of a recombinant Gluconobacter oxydans 621H strain (IK003.1) engineered for improved growth. Therefore we combined accurate Illumina sequencing technology and low-cost single-molecule nanopore sequencing using the MinION device from Oxford Nanopore. In our hybrid approach with a modified library protocol we could increase the average size of nanopore 2D reads to about 18.9kb. Combining the long MinION nanopore reads with the high quality short Illumina reads enabled the assembly of the engineered chromosome into a single contig and comprehensive detection and clarification of 7 structural variants including all three known genetically engineered modifications. We found the genome of IK003.1 was stable over 70 generations of strain handling including 28h of process time in a bioreactor. The long read data revealed a novel 1420 bp transposon-flanked and ORF-containing sequence which was hitherto unknown in the G. oxydans 621H reference. Further analysis and genome sequencing showed that this region is already present in G. oxydans 621H wild-type strains. Our data of G. oxydans 621H wild-type DNA from different resources also revealed in 73 annotated coding sequences about 91 uniform nucleotide differences including InDels. Together, our results contribute to an improved high quality genome reference for G. oxydans 621H which is available via ENA accession PRJEB18739.
高通量 DNA 测序技术的最新进展和新颖技术为生命科学带来了令人兴奋的机会和应用，包括微生物基因组学。短而高质量的读取数据不仅可以实现微生物基因组测序，而且在基因组组装和结构变异分析方面还不够充分，特别是在工程化的微生物细胞工厂中。产生长读长的单分子实时测序技术有望解决此类组装问题。在我们的研究中，我们希望用 R9 化学提高长纳米孔读取的平均读长，并采用混合方法分析结构变异，以检查为提高生长而工程化的氧化葡萄糖酸杆菌 621H 菌株（IK003.1）的基因组稳定性。因此，我们将准确的 Illumina 测序技术与使用牛津纳米孔 MinION 设备的低成本单分子纳米孔测序相结合。在我们的混合方法中，采用经过修改的文库方案，我们可以将纳米孔 2D 读取的平均大小增加到约 18.9kb。将长 MinION 纳米孔读取与高质量短 Illumina 读取相结合，使工程化染色体能够组装成单个连续序列，并全面检测和澄清包括所有三种已知基因工程修饰的 7 种结构变异。我们发现，IK003.1 的基因组在经过 70 代的菌株处理（包括在生物反应器中 28 小时的过程时间）后仍然稳定。长读数据揭示了一个新的 1420bp 转座子侧翼和包含 ORF 的序列，这在氧化葡萄糖酸杆菌 621H 参考基因组中是未知的。进一步的分析和基因组测序表明，该区域已经存在于氧化葡萄糖酸杆菌 621H 野生型菌株中。我们从不同资源获得的氧化葡萄糖酸杆菌 621H 野生型 DNA 的数据还显示，在 73 个注释编码序列中，大约有 91 个均匀的核苷酸差异，包括插入缺失。总之，我们的结果为氧化葡萄糖酸杆菌 621H 提供了一个改进的高质量基因组参考，该参考可通过 ENA 访问号 PRJEB18739 获得。
