Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang 310058, China.
Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang 310058, China;
Genome Res. 2023 Mar;33(3):412-426. doi: 10.1101/gr.277213.122. Epub 2023 Mar 23.
Tn5 transposon tagments double-stranded DNA and RNA/DNA hybrids to generate nucleic acids that are ready to be amplified for high-throughput sequencing. The nucleic acid substrates for the Tn5 transposon must be explored to increase the applications of Tn5. Here, we found that the Tn5 transposon can transpose oligos into the 5' end of single-stranded DNA longer than 140 nucleotides. Based on this property of Tn5, we developed a tagmentation-based and ligation-enabled single-stranded DNA sequencing method called TABLE-seq. Through a series of reaction temperature, time, and enzyme concentration tests, we applied TABLE-seq to strand-specific RNA sequencing, starting with as little as 30 pg of total RNA. Moreover, compared with traditional dUTP-based strand-specific RNA sequencing, this method detects more genes, has a higher strand specificity, and shows more evenly distributed reads across genes. Together, our results provide insights into the properties of Tn5 transposons and expand the applications of Tn5 in cutting-edge sequencing techniques.
Tn5 转座子将双链 DNA 和 RNA/DNA 杂种双链化，生成可用于高通量测序的扩增核酸。为了增加 Tn5 的应用，必须探索 Tn5 的核酸底物。在这里，我们发现 Tn5 转座子可以将寡核苷酸转座到长于 140 个核苷酸的单链 DNA 的 5'端。基于 Tn5 的这一特性，我们开发了一种基于标签化和连接的单链 DNA 测序方法，称为 TABLE-seq。通过一系列反应温度、时间和酶浓度测试，我们将 TABLE-seq 应用于起始总 RNA 量低至 30pg 的链特异性 RNA 测序。此外，与传统基于 dUTP 的链特异性 RNA 测序相比，该方法检测到更多的基因，具有更高的链特异性，并且在基因间表现出更均匀分布的读数。总之，我们的结果提供了对 Tn5 转座子特性的深入了解，并扩展了 Tn5 在尖端测序技术中的应用。
