Vaccine and Infectious Disease Organization (VIDO), University of Saskatchewan, Saskatoon, SK, Canada.
Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK, Canada.
BMC Genomics. 2023 Nov 21;24(1):698. doi: 10.1186/s12864-023-09784-6.
Avian pathogenic Escherichia coli (APEC) are the causative agents of colibacillosis in chickens, a disease which has significant economic impact on the poultry industry. Large plasmids detected in APEC are known to contribute to strain diversity for pathogenicity and antimicrobial resistance, but there could be other plasmids that are missed in standard analysis. In this study, we determined the impact of sequencing and assembly factors for the detection of plasmids in an E. coli whole genome sequencing project.
Hybrid assembly (Illumina and Nanopore) combined with plasmid DNA extractions allowed for detection of the greatest number of plasmids in E. coli, as detected by MOB-suite software. In total, 79 plasmids were identified in 19 E. coli isolates. Hybrid assemblies were robust and consistent in quality regardless of sequencing kit used or if long reads were filtered or not. In contrast, long read only assemblies were more variable and influenced by sequencing and assembly parameters. Plasmid DNA extractions allowed for the detection of physically smaller plasmids, but when averaged over 19 isolates did not significantly change the overall number of plasmids detected.
Hybrid assembly can be reliably used to detect plasmids in E. coli, especially if researchers are focused on large plasmids containing antimicrobial resistance genes and virulence factors. If the goal is comprehensive detection of all plasmids, particularly if smaller sized vectors are desired for biotechnology applications, the addition of plasmid DNA extractions to hybrid assemblies is prudent. Long read sequencing is sufficient to detect many plasmids in E. coli, however, it is more prone to errors when expanded to analyze a large number of isolates.
禽致病性大肠杆菌（APEC）是导致鸡大肠杆菌病的病原体，这种疾病对家禽业有重大的经济影响。在 APEC 中检测到的大型质粒被认为有助于致病性和抗菌抗性的菌株多样性，但可能还有其他在标准分析中遗漏的质粒。在这项研究中，我们确定了测序和组装因素对大肠杆菌全基因组测序项目中质粒检测的影响。
Illumina 和 Nanopore 的混合组装（hybrid assembly）加上质粒 DNA 的提取，通过 MOB-suite 软件检测到大肠杆菌中最多数量的质粒。在 19 个大肠杆菌分离株中总共鉴定出 79 个质粒。混合组装无论使用的测序试剂盒如何，或者是否过滤了长读长，其质量都很稳健且一致。相比之下，只有长读长的组装则更具多变性，并受测序和组装参数的影响。质粒 DNA 的提取允许检测到物理上较小的质粒，但在平均 19 个分离株中，并没有显著增加检测到的质粒总数。
混合组装可以可靠地用于检测大肠杆菌中的质粒，特别是如果研究人员专注于含有抗菌抗性基因和毒力因子的大型质粒。如果目标是全面检测所有质粒，特别是如果需要较小尺寸的载体用于生物技术应用，则将质粒 DNA 提取添加到混合组装中是明智的。长读测序足以检测大肠杆菌中的许多质粒，但是，当扩展到分析大量分离株时，更容易出错。
