不同样本前处理方法对高通量测序检测HBV、HCV、TTV结果的影响

Comparison of Different Sample Pretreatment Methods for Highthroughput Sequencing Detection Results of HBV, HCV and TTV

目的:探索不同样品前处理方法对于不同类型病毒检测的效果。方法:分别将携带乙肝病毒(双链环状DNA病毒)、丙肝病毒(单正链RNA病毒)、TTV(Torque teno virus)(单链环状DNA病毒)的血清等比例混合,模拟混合感染,然后分别采用多重置换扩增(MDA)和随机锚定PCR扩增并进行高通量测序,同时以原始样品(未扩增)直接高通量测序作为对照。结果:原始样品(未扩增)直接测序,产出的数据大部分为人类的序列;MDA方法中绝大部分数据为TTV、乙肝病毒;随机锚定PCR扩增方法中绝大部分数据为乙肝病毒。结论:MDA方法适合扩增环状病毒,随机锚定PCR扩增适合含量高的病毒,不做任何处理直接高通量测序检测病毒效果最差。本研究可为指导不同类型病原体如何选择扩增方案提供借鉴。

Objective： To explore the effect of different sample pretreatment methods for the detection of different types of virus. Methods： Three different kinds of virus serum, HBV, HCV and Torque teno virus（TTV）, were mixed under equal proportion to simulating of mixed infection. Mixed samples were amplified by multiple displacement amplification（MDA） and random anchor PCR amplification and then performing high throughput sequencing, at the meantime, a non-amplified mixed sample was sequenced as control. Results： The output data of non-amplified mixed sample were mostly human sequences; the sequencing data of MDA method were mostly HBV and TTV sequences; the generated data of random anchor PCR amplification were mostly HBV sequences. Conclusion：MDA method is appropriate for amplification of circular virus, random anchor PCR amplification is suit for sample with high level of virus, the worst method is directly high-throughput sequencing without any treatment. This study provides guidance for choosing amplification scheme when high-throughput sequencing different types of pathogens.