Abstract: Objective To establish a high-throughput semi-automatic bacterial whole-genome sequencing system to meet the needs of bacterial pathogen surveillance based on the genome sequence. Methods An automated liquid handler, disposable mechanical pipette tips, a 96-well nucleic acid extraction kit, an automated sequencing library construction system, and a sequencer were used to realize the semi-automation of bacterial genome sequencing. Results The distance between the mechanical pipette tip and the bottom of the sample well, the mixing speed of liquid, and the volume ratio of the mixed liquid to the sample each time were closely related to the cross-contamination of the samples. When the distance between the mechanical pipette tip and the bottom of the sample well was 2 mm, the mixing speed of liquid was 15% of the full speed, and the volume ratio of the mixed liquid to the sample was 50% each time, the contamination rate was 0. Compared with the manual procedure, this high-throughput semi-automatic bacterial whole-genome sequencing system improved the work efficiency by 4-fold. Conclusion A high-throughput semi-automatic bacterial whole-genome sequencing system is established, which can meet the needs of bacterial pathogen surveillance based on the genome sequence.

Key words: Automated liquid handling, Cross-contamination, Whole-genome sequencing, Pathogen surveillance network

摘要： 目的 建立高通量、半自动化细菌全基因组序列测定体系，满足基于基因组序列的细菌性病原体监测的需求。方法 使用自动化液体处理器、可丢弃性机械手枪头、96孔板型核酸提取试剂盒、自动测序文库构建系统以及测序仪，实现细菌基因组序列测定的半自动化。结果 机械手枪头与加样孔底的距离、液体混合速度和每次混匀液体的体积比3个因素与样品之间的交叉污染密切相关，机械手枪头与加样孔底的距离为2 mm、液体混合速度为全速的15%、每次混匀液体的体积比为50%时污染率为0。与手工方法相比，高通量半自动化细菌全基因组序列测定体系的工作效率提高了4倍。结论 建立的高通量、半自动化细菌基因组序列测定体系，能够满足基于基因组序列的细菌性病原体监测的需求。

关键词: 自动化液体处理, 交叉污染, 全基因组序列测定, 病原体网络化监测