Next Generation Sequencing Core, The Scripps Research Institute, La Jolla, CA 92040, USA.
Biotechniques. 2011 Mar;50(3):177-80. doi: 10.2144/000113613.
In this study, we tested the NuGEN Ovation RNA-Seq System for library preparation followed by next-generation sequencing on an Illumina GAIIx. The cDNA product of the NuGEN kit may have significant amounts of ssDNA with hairpin structures that are generated during the amplification process. These structures interfere with efficient downstream end repair, A-tailing, and adapter ligation, all standard steps in post-amplification sequencing library construction. We were able to increase the efficiency of sequencing library yields 4- to 6-fold or greater by treatment of NuGEN-amplified cDNA product with the single-strand endonuclease S1. These results suggest that this treatment effectively cleaves hairpin structures generated during amplification that are resistant to the standard enzyme cocktails used for the end-repair step.
在这项研究中，我们测试了 NuGEN Ovation RNA-Seq 系统，用于在 Illumina GAIIx 上进行文库制备和下一代测序。NuGEN 试剂盒的 cDNA 产物可能含有大量具有发夹结构的 ssDNA，这些结构是在扩增过程中产生的。这些结构会干扰下游末端修复、A 尾添加和接头连接等扩增后测序文库构建的标准步骤。我们通过用单链内切酶 S1 处理 NuGEN 扩增的 cDNA 产物，将测序文库的产量提高了 4 到 6 倍或更高。这些结果表明，这种处理可以有效地切割在扩增过程中产生的发夹结构，这些结构对末端修复步骤中使用的标准酶混合物具有抗性。
