高通量RNA测序(RNA-seq)极大地促进了我们对基因表达和疾病病因的理解,并且是识别各种生物体中生物标志物的强大工具。然而,大多数RNA-SEQ方法依赖于逆转录病毒逆转录酶(RT),具有固有的低保真度和持续合成能力的酶,将RNA转化为cDNA用于测序。这里,我们描述了使用热稳定II组内含子逆转录酶(TGIRT)的RNA-seq方案,具有高保真度,可持续发展,和链置换活动,以及有效的模板转换活性,可实现高效和无缝的RNA-seq衔接子添加。通过结合这些活动,TGIRT-seq能够同时分析来自少量起始材料的所有RNA生物型,具有优越的RNA-SEQ指标,以及前所未有的结构化RNA测序能力。Illumina测序的TGIRT-seq协议由三个步骤组成:(i)添加3'RNA-seq衔接子,与靶RNA的3'末端的cDNA合成的起始相结合,通过从合成衔接子RNA/DNA起始双链体的模板转换;(ii)添加5'RNA-seq衔接子,通过使用热稳定的5'AppDNA/RNA连接酶将衔接子寡核苷酸连接到完成的cDNA的3'末端;(iii)最小的PCR扩增,添加Illumina测序的捕获位点和索引。Illumina测序平台的TGIRT-seq已用于对核糖耗尽的编码和非编码RNA进行全面分析,化学片段化的细胞RNA,和分析完整的(非化学片段)细胞,细胞外囊泡(EV),和血浆RNA,它产生连续全长的结构小的非编码RNA(sncRNA)的端到端序列,包括tRNA,snoRNAs,snRNAs,pre-miRNA,和全长切除的线性内含子(FLEXI)RNA。图形摘要:图1。Illumina测序的TGIRT-seq方案概述。主要步骤是:(1)从具有1-nt3\'DNA突出端的合成R2RNA/R2RDNA起始双链体进行模板转换(A的混合物,C,G,和T残基,表示为N),与靶RNA的3个核苷酸碱基配对,通过添加dNTPs启动逆转录后,将R2R衔接子无缝连接到所得cDNA的5'末端;(2)将R1R衔接子连接到完成的cDNA的3'末端;和(3)使用添加Illumina捕获位点(P5和P7)和条形码序列(索引5和7)的引物进行最小PCR扩增。索引7条形码是必需的,虽然索引5条形码是可选的,提供独特的双指数(UDI)。
High-throughput RNA sequencing (RNA-seq) has extraordinarily advanced our understanding of gene expression and disease etiology, and is a powerful tool for the identification of biomarkers in a wide range of organisms. However, most RNA-seq methods rely on retroviral reverse transcriptases (RTs), enzymes that have inherently low fidelity and processivity, to convert RNAs into cDNAs for sequencing. Here, we describe an RNA-seq protocol using Thermostable Group II Intron Reverse Transcriptases (TGIRTs), which have high fidelity, processivity, and strand-displacement activity, as well as a proficient template-switching activity that enables efficient and seamless RNA-seq adapter addition. By combining these activities, TGIRT-seq enables the simultaneous profiling of all RNA biotypes from small amounts of starting material, with superior RNA-seq metrics, and unprecedented ability to sequence structured RNAs. The TGIRT-seq protocol for Illumina sequencing consists of three steps: (i) addition of a 3\' RNA-seq adapter, coupled to the initiation of cDNA synthesis at the 3\' end of a target RNA, via template switching from a synthetic adapter RNA/DNA starter duplex; (ii) addition of a 5\' RNA-seq adapter, by using thermostable 5\' App DNA/RNA ligase to ligate an adapter oligonucleotide to the 3\' end of the completed cDNA; (iii) minimal PCR amplification, to add capture sites and indices for Illumina sequencing. TGIRT-seq for the Illumina sequencing platform has been used for comprehensive profiling of coding and non-coding RNAs in ribodepleted, chemically fragmented cellular RNAs, and for the analysis of intact (non-chemically fragmented) cellular, extracellular vesicle (EV), and plasma RNAs, where it yields continuous full-length end-to-end sequences of structured small non-coding RNAs (sncRNAs), including tRNAs, snoRNAs, snRNAs, pre-miRNAs, and full-length excised linear intron (FLEXI) RNAs. Graphic abstract: Figure 1.Overview of the TGIRT-seq protocol for Illumina sequencing.Major steps are: (1) Template switching from a synthetic R2 RNA/R2R DNA starter duplex with a 1-nt 3\' DNA overhang (a mixture of A, C, G, and T residues, denoted N) that base pairs to the 3\' nucleotide of a target RNA, and upon initiating reverse transcription by adding dNTPs, seamlessly links an R2R adapter to the 5\' end of the resulting cDNA; (2) Ligation of an R1R adapter to the 3\' end of the completed cDNA; and (3) Minimal PCR amplification with primers that add Illumina capture sites (P5 and P7) and barcode sequences (indices 5 and 7). The index 7 barcode is required, while the index 5 barcode is optional, to provide unique dual indices (UDIs).