Xu Hengyi, Nottingham Ryan M, Lambowitz Alan M
Departments of Molecular Biosciences and Oncology, University of Texas at Austin, Austin, Texas, 78712, USA.
Bio Protoc. 2021 Dec 5;11(23):e4239. doi: 10.21769/BioProtoc.4239.
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).
高通量RNA测序（RNA-seq）极大地推动了我们对基因表达和疾病病因的理解，是在广泛生物体中鉴定生物标志物的强大工具。然而，大多数RNA-seq方法依赖逆转录病毒逆转录酶（RTs），这种酶固有地具有低保真度和持续合成能力，用于将RNA转化为cDNA进行测序。在此，我们描述了一种使用热稳定II型内含子逆转录酶（TGIRTs）的RNA-seq方案，TGIRTs具有高保真度、持续合成能力和链置换活性，以及高效的模板转换活性，能够实现高效且无缝的RNA-seq接头添加。通过结合这些活性，TGIRT-seq能够从少量起始材料同时分析所有RNA生物类型，具有卓越的RNA-seq指标，以及前所未有的对结构化RNA进行测序的能力。用于Illumina测序的TGIRT-seq方案包括三个步骤：（i）通过从合成接头RNA/DNA起始双链体进行模板转换，在目标RNA的3'端添加3' RNA-seq接头并启动cDNA合成；（ii）使用热稳定的5' App DNA/RNA连接酶将接头寡核苷酸连接到已完成cDNA的3'端，添加5' RNA-seq接头；（iii）进行最少的PCR扩增，以添加Illumina测序的捕获位点和索引。用于Illumina测序平台的TGIRT-seq已用于对核糖体去除、化学片段化的细胞RNA中的编码和非编码RNA进行全面分析，以及用于分析完整（非化学片段化）的细胞、细胞外囊泡（EV）和血浆RNA，在此过程中它能够产生结构化小非编码RNA（sncRNA）的连续全长端到端序列，包括tRNA、snoRNA、snRNA、前体miRNA和全长切除线性内含子（FLEXI）RNA。图形摘要：图1.Illumina测序的TGIRT-seq方案概述。主要步骤为：（1）从具有1个核苷酸3' DNA突出端（A、C、G和T残基的混合物，记为N）的合成R2 RNA/R2R DNA起始双链体进行模板转换，该突出端与目标RNA的3'核苷酸碱基配对，通过添加dNTPs启动逆转录后，将R2R接头无缝连接到所得cDNA的5'端；（2）将R1R接头连接到已完成cDNA的3'端；（3）使用添加Illumina捕获位点（P5和P7）和条形码序列（索引5和7）的引物进行最少的PCR扩增。索引7条形码是必需的，索引5条形码是可选的，以提供独特的双索引（UDI）。
