人恶性胸膜间皮瘤DNA从头测序及基因突变分析

DNADe Novo Sequencing and Gene MutationAnalysis ofHumanMalignant Pleural Mesothelioma①

通过DNA从头测序分析人胸膜间皮瘤发生的高关联度突变基因。提取恶性胸膜间皮瘤(MPM)组织和正常胸膜组织DNA,构建基因文库,用Illumina HiSeqX Ten PE 150平台测序,将测序结果与人类基因组数据库的参考序列进行比对、注释,并对测序结果进行过滤、错误率分布检查、GC含量分布检查分析。MPM组织DNA平均过滤37829946 bp,错误率小于0.12%,GC含量占41.17%,而正常胸膜组织DNA平均过滤39089681 bp,错误率小于0.1%,GC含量占41.7%,两者测序质量均在Q 30(≥80%)以上,MPM为87.43%,正常胸膜为88.36%。以上高质量测序数据通过BWA比对到参考基因组(GRCh 37/hg 19),得到最初比对序列,利用重复标记后的比对序列进行覆盖度、深度等统计,覆盖深度达到10 X以上该突变位点可信。结果显示,实验病例XL14覆盖深度达到10 X的占98.59%,覆盖率达到99.83%;对照病例Z5占98.50%,覆盖率达到99.79%。对该序列进行基因注释分析,发现一系列单核苷酸多态性、基因插入缺失、基因结构变异、基因拷贝数变异,筛选出总变异位点数29277个,可能致病的变异位点数22个,致病性的变异位点数5个,不确定变异有害性的位点数为3353个,其余变异位点均为良性。进一步对突变基因进行富集、关联性分析,预测出突变基因TXNDC2与人胸膜间皮瘤的发生高度相关,相关系数达到0.8以上;突变基因PIEN、ABCC1、UGT1A7、UGT1A3、UGT1A4、UGT1A9、ALDH3B1、UGT1A5等与人胸膜间皮瘤有一定关联性,关联度在0~0.2之间。基因TXNDC2、PIEN、ABCC1、UGT1A7、UGT1A3、UGT1A4、UGT1A9、ALDH3B1、UGT1A5的变异可能与人胸膜间皮瘤的发生发展有关。本实验为人胸膜间皮瘤分子诊断提供了参考。

To analyze the high correlation mutation genes of human pleural mesothelioma by DNA de novo sequencing,DNA was extracted from malignant pleural mesothelioma(MPM)tissues and normal pleural tissues,and a gene library was constructed.The Illumina HiSeqX Ten PE 150 platform was used for sequencing.The sequencing results were compared and annotated with the reference sequence of the human genome database.The sequencing results were filtered,the error rate distribution was checked,and the GC content distribution was checked and analyzed.The average filtering of MPM tissue DNA was 37829946 bp,the error rate was less than 0.12%,and the GC content accounted for 41.17%.The average filtering of normal pleural tissue DNA was 39089681 bp,the error rate was less than 0.1%,and the GC content accounted for 41.7%.The sequencing quality of both was above Q30(≥80%),MPM was 87.43%,and normal pleura was 88.36%.The above high-quality sequencing data were compared to the reference genome(GRCh 37/hg19)by BWA,and the initial alignment sequence was obtained.The coverage and depth of the alignment sequence after repeated labeling were used for statistics.The mutation site was credible when the coverage depth reached 10 X or more.The results showed that 98.59%of the experimental cases XL14 had a coverage depth of 10 X,and the coverage rate reached 99.83%.The control case Z5 accounted for 98.50%,and the coverage rate reached 99.79%.Gene annotation analysis of the sequence revealed a series of single nucleotide polymorphisms,gene insertion and deletion,gene structure variation,and gene copy number variation.The total number of variation sites was 29277,and the number of possible pathogenic variation sites was 22.The number of pathogenic variation sites was 5,the number of sites with uncertain variation was 3353,and the remaining variation sites were benign.Further enrichment and correlation analysis of mutant genes were carried out to obtain the correlation map and predict the mutation.