Transposase-assisted tagmentation of RNA/DNA hybrid duplexes

Abstract

Tn5-mediated transposition of double-strand DNA has been widely utilized in various high-throughput sequencing applications. Here, we report that the Tn5 transposase is also capable of direct tagmentation of RNA/DNA hybrids in vitro. As a proof-of-concept application, we utilized this activity to replace the traditional library construction procedure of RNA sequencing, which contains many laborious and time-consuming processes. Results of Transposase assisted RNA/DNA hybrids Co-tagmEntation (termed 'TRACE-seq') are compared to traditional RNA-seq methods in terms of detected gene number, gene body coverage, gene expression measurement, library complexity, and differential expression analysis. At the meantime, TRACE-seq enables a cost-effective one-tube library construction protocol and hence is more rapid (within 6h) and convenient. We expect this tagmentation activity on RNA/DNA hybrids to have broad potentials on RNA biology and chromatin research.

Genomic profiling of native R loops with a DNA-RNA hybrid recognition sensor

“…Tn5 has been widely used in sequencing library preparation for rapid processing and low sample input requirement of dsDNA. In addition to its canonical function in dsDNA tagmentation, Tn5 transposase was recently shown to bind and effectively transpose both strands of DNA-RNA hybrids, which can be amplified for library preparation after strand displacement, avoiding many time-consuming steps ( 30 , 31 ). Since Tn5 transposase does not react with ssRNA or ssDNA, there are no concerns with the possible RNA contamination or the off-target effect of HBD or S9.6 at loci with ssDNA.…”

“…HBD mediates the specific recognition of DNA-RNA hybrids in a nonsequence-specific manner ( 22 ), which highlights the domain itself as a potential sensor for R loop mapping. Recently, Tn5 transposase was reported to randomly bind DNA-RNA hybrids and transpose adapters onto both strands of the DNA-RNA hybrids ( 30 , 31 ). Besides, the transposed products could have the strand displaced and be directly used for sequencing library preparation, which save many time-consuming steps ( 30 , 31 ).…”

“…Recently, Tn5 transposase was reported to randomly bind DNA-RNA hybrids and transpose adapters onto both strands of the DNA-RNA hybrids ( 30 , 31 ). Besides, the transposed products could have the strand displaced and be directly used for sequencing library preparation, which save many time-consuming steps ( 30 , 31 ). Using the Tn5 system for DNA and DNA-RNA hybrids tagmentation would potentially avoid fragmentation bias caused by restriction enzyme digestion.…”

High‐Throughput Metagenomics for Identification of Pathogens in the Clinical Settings

“…Compared to existing methods, it significantly simplifies the process of database construction and reduces the initial template input, which is relevant for samples with low starting concentrations, including that of coronaviruses. [ 60,61 ]…”

Library Preparation Based on Transposase Assisted RNA/DNA Hybrid Co-Tagmentation for Next-Generation Sequencing of Human Noroviruses

“…DNA sequencing library preparation based on Tn5 transposase could be processed conveniently and has low requirements for sample inputs [ 16 , 17 ]. Tn5 transposase has been altered to be capable of direct tagmentation of RNA/DNA hybrids in vitro in 2020, named Transposase-assisted RNA/DNA hybrids Co-tagmEntation (‘TRACE-seq’) [ 18 , 19 ]. TRACE-seq offers a greatly simplified protocol and produces results with higher reproducibility and GC uniformity compared with prevailing RNA-seq methods [ 18 ].…”