Integration activity of evolutionarily recent human retroelements Alu and LINE in tumors

The activity of mobile elements can be considered as one of the aspects of tumor genomic instability. From that perspective, it has a potential as a prognostic or diagnostic marker. In this work we attempted to assess the levels of integration activity of evolutionarily recent human retroelements (RE) in tumors and their relationship with RE transcriptional activity as well as other potential regulatory factors.To determine the integration activity of RE in tumors and to assess its correlation with RE transcriptional activity we performed a genome-wide search for tumor-specific inserts. For that we selected 20 tumor samples with higher and 20 samples with lower LINE L1 L1Hs and L1PA2 expression for further sequencing of genomic flanks of L1Hs L1PA2 and AluYa5/8 elements. Corresponding matched norm samples were used as a control. Overall design: All experimental biosamples were FFPE tumor tissue blocks. Histological materials were assessed by a pathologist and selected areas with at least 50% of tumor cells were dissected for further nucleic acids extraction. In some cases, adjacent non-tumour tissue was also marked and dissected. Total RNA was extracted from FFPE samples with RNeasy FFPE Kit (Qiagen) following the manufacturer's protocol. For depletion of ribosomal RNA and library preparation, the KAPA RNA HyperPrep Kit with RiboErase (HMR) (Roche) was used. KAPA single-indexed adaptors (Roche) or KAPA unique dual indexed primers (Roche) were used for multiplexing samples in one sequencing run. Sequencing was performed on Illumina NextSeq 550(Illumina) with equipment for single-end 75-bp read length sequencing, for approximately 30 million raw reads per sample.可移动遗传元件的活性可被视为肿瘤基因组不稳定性的重要研究维度之一。从该视角出发，其具备作为预后或诊断标志物的潜在价值。本研究旨在评估进化上较为年轻的人类反转录转座子（retroelements, RE）在肿瘤中的整合活性水平，及其与反转录转座子转录活性以及其他潜在调控因子的关联。 为确定肿瘤中反转录转座子的整合活性并评估其与反转录转座子转录活性的相关性，我们开展了全基因组范围的肿瘤特异性插入序列搜寻工作。为此，我们筛选出20例LINE L1、L1Hs及L1PA2表达水平较高的肿瘤样本，以及20例表达水平较低的样本，用于后续对L1Hs、L1PA2及AluYa5/8元件的基因组侧翼序列进行测序。相应的配对正常组织样本作为对照。 整体实验设计如下：所有实验生物样本均为福尔马林固定石蜡包埋（Formalin-Fixed Paraffin-Embedded, FFPE）肿瘤组织块。病理学家对组织学标本进行评估，选取肿瘤细胞占比至少50%的区域进行解剖切割，用于后续核酸提取。部分病例中，邻近的非肿瘤组织也会被标记并切割取样。总RNA提取采用RNeasy FFPE试剂盒（Qiagen），严格遵循厂商提供的实验流程。核糖体RNA去除及文库制备环节使用了带有RiboErase (HMR)的KAPA RNA HyperPrep试剂盒（Roche）。实验采用KAPA单索引接头（Roche）或KAPA独特双索引引物（Roche），以实现在单次测序运行中对多个样本进行多重测序。测序工作在Illumina NextSeq 550（Illumina）平台上完成，采用单端75bp读长的测序模式，每个样本约获得3000万条原始测序读段。