痕量样本高通量测序文库构建

Construction of high throughput sequencing library for trace samples

中文关键词:  高通量测序  文库构建  微生物  痕量

英文关键词:high throughput sequencing  library construction  microorganism  trace

中文摘要:

      目的 通过调整建库过程中的测序接头加入量、片段选择的执行与否、文库扩增循环数等条件, 建立一种基于Illumina测序平台的高效稳定的痕量样本高通量文库构建方法。方法 采用不同类型的样本, 设计不同起始DNA量(最低至0.1 ng)的文库构建策略, 主要对接头使用量、扩增循环数等进行优化, 并通过数据分析评估文库质量。结果 对于起始量低至0.1 ng的DNA样本, 基因组文库构建成功率达100%。对于基因组较小的细菌, 0.1 ng的起始DNA量的数据组装效果与100 ng起始DNA量相近; 对于基因组较大、结构复杂的细菌, 0.1 ng的起始DNA量的数据组装效果显著差于100 ng起始DNA量, 但可通过增加测序数据量的方法提高组装效果。对于元基因组测序, 起始DNA量为1 ng时, 制备的测序文库与常规起始DNA量制备的文库得到的微生物群落结构相近。结论 本研究建立的0.1 ng起始DNA量文库构建方法稳定, 可用于更多微生物基因组测序、元基因组测序的应用领域。

英文摘要:

      Objective To establish an efficient and stable high-throughput library construction method of trace samples based on Illumina sequencing platform by adjusting the amount of sequencing adapter, the execution of fragment selection, the number of library amplification cycles and so on. Methods Different types of samples were used to design the construction strategy of library with different initial DNA amount (as low as 0.1 ng). The usage of adapter and the number of amplification cycles were optimized, and the quality of library was evaluated by data analysis. Results The success rate of genomic library construction was 100% for DNA samples with the initial amount as low as 0.1 ng. For bacteria with small genome, the data assembly effect of 0.1 ng initial DNA was similar to that of 100 ng initial DNA. For bacteria with large genome and complex structure, the data assembly effect of 0.1 ng initial DNA was significantly worse than that of 100 ng initial DNA, but the assembly effect could be improved by increasing the amount of sequencing data. For metagenomic sequencing, when the initial DNA quantity was 1 ng, the microbial community structure of the library was similar to that of the library with conventional initial DNA quantity. Conclusion The construction method of 0.1 ng initial DNA library established in this study is stable and can be used in more application fields of microbial genome sequencing and metagenome sequencing.