Ordered two-template relay for automation-friendly, low-bias, end-to-end RNA sequence inventories of complex ncRNA samples

NGS libraries from sRNA are commonly prepared by sequential ligation of adaptors to input RNA 3′ and 5′ ends, often with a gel purification step after each ligation step, followed by cDNA synthesis and PCR. Ligase bias in miRNA capture is reduced by the use of degenerate sequence (“4N”) at adaptor ends. However, 4N protocols are time consuming in manual effort, technically challenging, and require high-input RNA amount due to many steps with product loss. Researchers at the University of California, Berkeley sought to exploit the serial template jumping ability of BoMoC reverse transcriptase as the basis of a ligation-independent method for end-to-end sRNA sequence capture into NGS libraries. Ultimately, they developed Ordered Two-Template Relay (OTTR): a single-tube reverse transcription reaction for dual-end adaptor-tagged cDNA library synthesis.

OTTR for NGS cDNA library generation

(A) Optimized workflow for single-tube synthesis of cDNA libraries. A pool of RNA and/or DNA input molecules (maroon) is first labeled by BoMoC with 3′ ddRTP. Buffer conditions are then toggled from Mn²⁺ to Mg²⁺ and any free ddRTPs are inactivated. Next, dNTPs, oligonucleotides, and BoMoC are added to initiate cDNA synthesis from the RNA-DNA primer duplex across the IT (maroon), ending after copying the AT (green). If desired, products can then be treated with RNase A and RNase H to remove RNA, yielding the desired cDNA. (B) Schematic of primers involved in Illumina Full-length (Top) or Universal (Bottom) adaptor addition and their respective cDNA library products. DNA primers were the complement of P7-i7-R2 or R2, while ATs were P5-i5-R1 or R1. In the Full-length adaptor strategy, only cDNA products elongated by copying the AT can bind to the flow cell. The covalently linked blocking group is indicated by a diamond.