Transcriptome sequencing of human hepatocellular carcinoma. Homo sapiens

Deep high-throughput transcriptome sequencing (RNA-seq) performed on 3 pairs of matched tumor and adjacent non-tumorours (NT) tissues from HCC patients of Chinese origin generated 183.6-million reads that could be aligned. We discovered a number of differentially expressed genes and multiple types of somatic single nucleotide variations (SNVs) in expressed genes. After the removal of the error alignments, high-quality reads were mapped to the human reference sequence (GRCh37/hg19) using three different softwares TopHat, Burrows-Wheeler Aligner (BWA) and CLC Genomics Workbench (CLC). The high-quality variants were identified using VarScan with the following parameters: minimum coverage depth of 10, variation frequency of more than 30% and base quality of more than 15. A total of 568, 545 and 494 potential somatic single nucleotide variants (SNVs), including 94, 89 and 101 coding somatic SNVs (cSNVs), were identified in 3 tumor samples HCC448T, HCC473T and HCC510T, respectively. Validation analysis was carried out for 10 of the intersected cSNVs (all are non-synonymous substitutions) within selected genes of interests with the majority confirmed. Overall design: Examination of 3 paired human hepatocellular carcinoma and matched non-tumor tissues

本研究针对3对中国肝细胞癌（hepatocellular carcinoma, HCC）患者的匹配肿瘤及邻近正常（non-tumor, NT）组织开展深度高通量转录组测序（RNA-seq），共获得183.6百万条可比对读段。研究人员在表达基因中鉴定出一批差异表达基因与多种类型的体细胞单核苷酸变异（somatic single nucleotide variations, SNVs）。在剔除错误比对的读段后，使用TopHat、Burrows-Wheeler Aligner（BWA）与CLC Genomics Workbench（CLC）三款不同软件，将高质量读段比对至人类参考基因组序列GRCh37/hg19。基于VarScan软件，以最低测序深度10、变异频率超过30%、碱基质量值高于15作为参数，鉴定得到高质量变异位点。在HCC448T、HCC473T与HCC510T这3份肿瘤样本中，分别鉴定得到568、545和494个潜在体细胞单核苷酸变异（SNVs），其中包含94、89和101个编码区体细胞单核苷酸变异（coding somatic SNVs, cSNVs）。针对10个交集编码区体细胞单核苷酸变异（均为非同义替换）的目标候选基因开展验证分析，其中大部分变异得到了确认。整体实验设计：对3对配对的人类肝细胞癌及匹配的邻近正常组织进行检测。