Profiling gene expression changes in primary ovarian tumors compared to matched metastasis

Ovarian cancer is the deadliest gynecologic malignancy and the fifth leading cause for cancer related deaths among women in USA. Most patients have metastatic disease at the time of diagnosis and this causes the poor prognosis. For a comprehensive understanding of the gene expression changes accompanying ovarian cancer metastasis, we isolated RNA from 11 pairs of matched primary tumors and metastasis from OC patients and sequenced them. Library preparation and next generation RNA sequencing was carried out at the Center for Genomics and Bioinformatics core facility, Indiana University, Bloomington. The library preparation was done using TruSeq Stranded mRNA HT Sample Prep kit (Illumina cat#RS-122-2103) according to the manufacturer’s protocol and 8-neucleotide barcodes were added for multiplexing. The barcoded libraries were cleaned by bead cut with AMPure XP beads (Beckman Coulter, cat#A63882), verified using Qubit3 fluorometer (ThermoFisher Scientific) and 2200 TapeStation bioanalyzer (Agilent Technologies), and then pooled. The pool was sequenced on NextSeq 500 (Illumina) with NextSeq75 High Output v2 kit (Illumina cat#FC-404-2005). 11 pairs of matched primary ovarian tumors and metastasis from ovarian cancer patients were used for sequencing. The library preparation was done using TruSeq Stranded mRNA HT Sample Prep kit (Illumina cat#RS-122-2103) according to the manufacturer’s protocol and 8-neucleotide barcodes were added for multiplexing. The barcoded libraries were cleaned by bead cut with AMPure XP beads (Beckman Coulter, cat#A63882), verified using Qubit3 fluorometer (ThermoFisher Scientific) and 2200 TapeStation bioanalyzer (Agilent Technologies), and then pooled. The pool was sequenced on NextSeq 500 (Illumina) with NextSeq75 High Output v2 kit (Illumina cat#FC-404-2005). Reads were adapter trimmed and quality filtered using Trimmomatic ver. 0.33, with the cutoff threshold for average base quality score set at 20 over a sliding window of 3 bases. Reads shorter than 20 bases post-trimming were excluded. Cleaned reads mapped to human genome GRCh38 with gencode v25 annotation using Tophat2 ver 2.1.0. Significantly differentially expressed genes at 5% FDR with at least 2 fold change were called using DESeq2 ver. 1.12.3.

卵巢癌是致死性最高的妇科恶性肿瘤，同时也是美国女性癌症相关死亡的第五大诱因。多数患者在确诊时已发生转移性疾病，这也是其预后不佳的核心原因。为全面解析卵巢癌转移过程中伴随的基因表达变化，我们从11例卵巢癌（Ovarian Cancer，OC）患者的配对原发肿瘤与转移灶中分离RNA并进行测序。文库构建与下一代RNA测序工作由印第安纳大学布卢明顿分校基因组与生物信息学核心实验室完成。本次文库制备采用TruSeq Stranded mRNA HT样本制备试剂盒（Illumina，货号RS-122-2103），严格遵循厂商说明书操作流程，并添加8核苷酸条形码以实现多重测序。带条形码的文库经AMPure XP磁珠（Beckman Coulter，货号A63882）磁珠纯化，通过Qubit3荧光计（赛默飞世尔科技）与2200 TapeStation生物分析仪（安捷伦科技）完成质量验证后进行混合。混合后的文库使用NextSeq 500测序平台（Illumina）搭配NextSeq75 High Output v2试剂盒（Illumina，货号FC-404-2005）进行测序。本研究共纳入11例卵巢癌患者的配对原发卵巢肿瘤与转移灶样本用于测序，其文库制备流程为：采用TruSeq Stranded mRNA HT样本制备试剂盒（Illumina，货号RS-122-2103），严格按照厂商说明书操作，并添加8核苷酸条形码以实现多重测序。带条形码的文库经AMPure XP磁珠（Beckman Coulter，货号A63882）磁珠纯化，通过Qubit3荧光计（赛默飞世尔科技）与2200 TapeStation生物分析仪（安捷伦科技）完成质量验证后进行混合。混合后的文库使用NextSeq 500测序平台（Illumina）搭配NextSeq75 High Output v2试剂盒（Illumina，货号FC-404-2005）进行测序。测序得到的读段使用Trimmomatic 0.33版本进行接头修剪与质量过滤，设置滑动窗口大小为3个碱基，平均碱基质量评分阈值为20；修剪后长度小于20碱基的读段将被剔除。将清洁后的读段比对至带有gencode v25注释的人类基因组GRCh38，比对工具为Tophat2 2.1.0版本。采用DESeq2 1.12.3版本鉴定错误发现率（False Discovery Rate，FDR）校正后显著性水平≤5%且表达变化倍数≥2的显著差异表达基因。