背景:牛津纳米孔提供了高通量测序平台,能够以99.95%的准确性重建完整的细菌基因组。然而,即使是很小的错误也会掩盖密切相关的分离株之间的系统发育关系。已开发出抛光工具来纠正这些错误,但尚不确定他们是否获得高分辨率食源性疾病暴发源跟踪所需的准确性。
结果:我们测试了132种组装和短期和长期阅读抛光工具的组合,以评估其重建来自2020年洋葱爆发的15种高度相似的肠道沙门氏菌血清型Newport分离株基因组序列的准确性。虽然长读抛光单独提高了精度,接近完美的准确性(99.9999%的准确性或在4.8Mbp基因组中存在约5个核苷酸错误,排除低置信度区域)仅通过组合了长读数和短读数抛光工具的管道获得。值得注意的是,medaka是比Racon更准确,更有效的长读抛光机。在短读抛光机中,NextPolish显示了最高的准确性,但是Pilon,Polypolish,和POLCA的表现类似。在5个表现最好的管道中,用medaka抛光,其次是nextpolish是最常见的组合。重要的是,抛光工具的顺序很重要,即在更准确的工具引入错误之后,使用不太准确的工具。均聚物和重复区域的Indels,其中短读取不能唯一映射,仍然是最难纠正的错误。
结论:仍然需要短读数来纠正纳米孔测序组装中的错误,以获得源跟踪调查所需的准确性。我们对抛光管道性能的细粒度评估使我们能够为工具用户提供最佳实践,并为工具开发人员提供改进的领域。
BACKGROUND: Oxford Nanopore provides high throughput sequencing platforms able to reconstruct complete bacterial genomes with 99.95% accuracy. However, even small levels of error can obscure the phylogenetic relationships between closely related isolates. Polishing tools have been developed to correct these errors, but it is uncertain if they obtain the accuracy needed for the high-resolution source tracking of foodborne illness outbreaks.
RESULTS: We tested 132 combinations of assembly and short- and long-read polishing tools to assess their accuracy for reconstructing the genome sequences of 15 highly similar Salmonella enterica serovar Newport isolates from a 2020 onion outbreak. While long-read polishing alone improved accuracy, near perfect accuracy (99.9999% accuracy or ~ 5 nucleotide errors across the 4.8 Mbp genome, excluding low confidence regions) was only obtained by pipelines that combined both long- and short-read polishing tools. Notably, medaka was a more accurate and efficient long-read polisher than Racon. Among short-read polishers, NextPolish showed the highest accuracy, but Pilon, Polypolish, and POLCA performed similarly. Among the 5 best performing pipelines, polishing with medaka followed by NextPolish was the most common combination. Importantly, the order of polishing tools mattered i.e., using less accurate tools after more accurate ones introduced errors. Indels in homopolymers and repetitive regions, where the short reads could not be uniquely mapped, remained the most challenging errors to correct.
CONCLUSIONS: Short reads are still needed to correct errors in nanopore sequenced assemblies to obtain the accuracy required for source tracking investigations. Our granular assessment of the performance of the polishing pipelines allowed us to suggest best practices for tool users and areas for improvement for tool developers.