样本采集与处理条件对中华按蚊转录组测序结果的影响

INFLUENCES OF SAMPLING PROTOCOL AND PROCESSING CONDITIONS ON TRANSCRIPTOME SEQUENCING ANALYSES OF ANOPHELES SINENSIS

中华按蚊是严重威胁人类健康的害虫,对其生物样本进行转录组分析是风险评估与监测的常用重要手段。本文评估了吸血、存活状态及妥善存入RNA贮存液前的冷冻时间等变量对于中华按蚊转录组测序及分析结果的影响。研究共对4组12个中华按蚊野生样本进行测序,获得48775个基因及其对应表达量。各样本质量通过核心直系同源基因与检出Unigene完整性进行衡量,计算过程与结果数据均通过统计学显著性检测(Qvalue≤0.01)。结果显示,吸血活体且保存至RNA贮存液前未经冷冻的样本质量最佳,其核心直系同源基因与检出Unigene完整性均超过90%。非吸血样本组装结果呈碎片化,表现为整体检出基因比例高达94.07%,但完整核心直系同源基因比例却仅为63.3%。冷冻过程对转录组测序结果影响极为严重,无论个体冷冻前存活与否,20 min左右即可导致20%以上的基因缺失。本研究为评估中华按蚊转录组数据有效性,以及之后提升相关研究的准确性提供了理论基础与数据支持。

Anopheles sinensis is a kind of pest seriously threatening human health.Transcriptome analysis with its biological samples is an important method for risk assessment and monitoring.The effects were evaluated in present paper of blood sucked,status(dead/alive),and the freezing time before proper RNA storage on transcriptome sequencing and analysis results of A.sinensis.Four groups including 12 wild A.sinensis samples were sequenced,and 48775 genes and their corresponding expression levels were obtained.The quality of each sample was measured by the integrity of core homologous genes and detected Unigenes,and the calculation process and result data were all statistically significant(Q-value≤0.01).The results showed that the quality of live and blood-sucked samples not frozen before being stored in RNA storage solution was the best,and both the integrity of core homologous genes and Unigene detected exceeded 90%.The assembly results of non-bloodsucking samples appeared in fragmentation,showing that the overall proportion of genes detected reached 94.07%,but the proportion of intact core homologous genes was only 63.3%.The freezing process has a very serious impact on the results of transcriptomic sequencing,and more than 20%gene deletion can occur within about 20 minutes regardless of the survival of the individual before frozen.The present study provided a theoretical basis and data support for evaluating the validity of transcriptome data of Anopheles sinensis and improved the accuracy of related studies later.