Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
G3 (Bethesda). 2021 Jun 17;11(6). doi: 10.1093/g3journal/jkab083.
The newest generation of DNA sequencing technology is highlighted by the ability to generate sequence reads hundreds of kilobases in length. Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) have pioneered competitive long read platforms, with more recent work focused on improving sequencing throughput and per-base accuracy. We used whole-genome sequencing data produced by three PacBio protocols (Sequel II CLR, Sequel II HiFi, RS II) and two ONT protocols (Rapid Sequencing and Ligation Sequencing) to compare assemblies of the bacteria Escherichia coli and the fruit fly Drosophila ananassae. In both organisms tested, Sequel II assemblies had the highest consensus accuracy, even after accounting for differences in sequencing throughput. ONT and PacBio CLR had the longest reads sequenced compared to PacBio RS II and HiFi, and genome contiguity was highest when assembling these datasets. ONT Rapid Sequencing libraries had the fewest chimeric reads in addition to superior quantification of E. coli plasmids versus ligation-based libraries. The quality of assemblies can be enhanced by adopting hybrid approaches using Illumina libraries for bacterial genome assembly or polishing eukaryotic genome assemblies, and an ONT-Illumina hybrid approach would be more cost-effective for many users. Genome-wide DNA methylation could be detected using both technologies, however ONT libraries enabled the identification of a broader range of known E. coli methyltransferase recognition motifs in addition to undocumented D. ananassae motifs. The ideal choice of long read technology may depend on several factors including the question or hypothesis under examination. No single technology outperformed others in all metrics examined.
新一代 DNA 测序技术的特点是能够生成数百千碱基长的序列读取。Pacific Biosciences（PacBio）和 Oxford Nanopore Technologies（ONT）开创了具有竞争力的长读长平台，最近的工作重点是提高测序通量和每个碱基的准确性。我们使用三种 PacBio 方案（Sequel II CLR、Sequel II HiFi、RS II）和两种 ONT 方案（Rapid Sequencing 和 Ligation Sequencing）产生的全基因组测序数据，比较了细菌大肠杆菌和果蝇黑腹果蝇的组装。在测试的两种生物中，即使考虑到测序通量的差异，Sequel II 组装也具有最高的一致性准确性。与 PacBio RS II 和 HiFi 相比，ONT 和 PacBio CLR 测序的读取最长，组装这些数据集时基因组连续性最高。除了基于连接的文库外，ONT Rapid Sequencing 文库还具有最少的嵌合读取，并且可以对大肠杆菌质粒进行更好的定量。通过采用混合方法使用 Illumina 文库进行细菌基因组组装或对真核基因组组装进行抛光，可以提高组装质量，并且对于许多用户来说，ONT-Illumina 混合方法将更具成本效益。可以使用这两种技术检测全基因组 DNA 甲基化，但除了未记录的 D. ananassae 基序外，ONT 文库还能够识别更广泛的已知大肠杆菌甲基转移酶识别基序。长读长技术的理想选择可能取决于几个因素，包括正在检查的问题或假设。没有一种技术在所有检查的指标上都表现出色。
