高通量测序技术分析肺结核患者PBMC基因表达谱差异

摘要

Objective:The detection of PBMC expression profile between normal people and tuberculosis patients,and compare the gene expression difference between them using high throughput sequencing technique in order to seek tuberculosis morbidity related gene.Methods:The cDNA library built from the total RNA isolated from the PBMC separated from normal people and patients of tuberculosis were amplified using bridge PCR by fixing on Flow cell through adaptor sequence,and then sequenced using Illumina sequencer.The differential gene expression profile of the two populations was analyzed using bioinformatics methods.Results:12 270 genes of 36390 distinct tags from tuberculosis samples and 12 244 genes of 35 009 distinct tags from normal samples were obtained from the raw data generated by sequencing after filtering,alignment,and annotation.According to the standard of FDR ≤0.001,l log2 Ratiol ≥ 1,3 097 differential genes were screened with 1 601 up regulated and 1 469 down regulated.33 significant differentially expressed genes with 16 up regulated and 17 down regulated were screened by increasing the standard by 4 fold and controlling the expression level.Conclusion:Most differential genes located in cells act as connecting function (protein junction) with enzyme catalytic activity,played important roles in metabolism,and mainly in MAPK signal pathway as well as chemokine factor signal pathway.These differential genes can be used as references of diagnose markers and drug targets in the area of tuberculosis.%目的:应用Illumina高通量测序技术研究肺结核患者和健康人群PBMC基因表达谱差异,以寻找与肺结核发病相关的基因.方法:分离肺结核患者和健康人群PBMC,提取总RNA,并制备cDNA文库,通过接头序列固定在Flow cell上进行桥式PCR扩增,再用Illumina测序仪进行深度测序,结合生物信息方法分析两类人群基因表达谱的差异情况.结果:测序产生的原始数据经过滤、比对、注释后得到肺结核样本36 390类标签12 270个基因,正常对照样本35 009类标签12 244个基因.按照FDR≤0.001,l log2 Ratiol≥1筛选标准,筛选出3 097个差异基因,其中上调基因1 601个,下调基因1 469个.增大筛选标准至4倍,并对表达量进行控制,筛选出33个差异极显著基因,其中上调基因16个,下调基因17个.结论:差异基因大部分位于细胞内,起着连接功能(蛋白质连接),具有酶催化活性,在代谢中起着重要作用,并主要位于MAPK信号通路和趋化因子信号通路中.这些差异基因可以为今后肺结核领域筛选诊断标识和药物作用靶点做参考.