Gorbacheva Tatyana, Quispe-Tintaya Wilber, Popov Vasily N, Vijg Jan, Maslov Alexander Y
Department of Genetics, Albert Einstein College of Medicine, New York, NY.
Department of Genetics, Cytology, and Bioengineering, Voronezh State University, Voronezh, Russia.
Biotechniques. 2015 Apr 1;58(4):200-2. doi: 10.2144/000114277. eCollection 2015 Apr.
A transposon-based approach for the construction of sequencing libraries is an efficient way of preparing samples for processing on both Illumina and Ion Torrent platforms. However, PCR-mediated incorporation of adaptors in tagged DNA fragments leaves behind self-complementary regions flanking the DNA fragment. These regions are capable of forming hairpin structures and, together with adaptors, create conditions for the potential formation of template heteroduplexes. These negatively affect the sequencing process on the Ion Torrent platform and can lead to a more than 3-fold decline in output data compared with sequencing of conventional libraries. To address this problem, we have developed MuPlus, a transposon-based protocol for barcoded library preparation for Ion Torrent, in which one adaptor is integrated by PCR and the second is integrated by ligation as a single-stranded oligonucleotide after enzymatic cleavage of a complementary part on one strand of the tag. The resulting library does not contain self-complementary, hairpin-forming regions, is free of heteroduplexes, and can be analyzed on the Ion Torrent platform with the same efficiency as a library created with a ligation-based protocol.
一种基于转座子构建测序文库的方法是在Illumina和Ion Torrent平台上高效制备样本用于处理的方式。然而，PCR介导的接头掺入到带标签的DNA片段中会在DNA片段两侧留下自我互补区域。这些区域能够形成发夹结构，并与接头一起为模板异源双链体的潜在形成创造条件。这些对Ion Torrent平台上的测序过程产生负面影响，与传统文库测序相比，可能导致输出数据下降超过3倍。为了解决这个问题，我们开发了MuPlus，这是一种基于转座子的用于Ion Torrent条形码文库制备的方案，其中一个接头通过PCR整合，另一个接头在标签一条链上的互补部分酶切后作为单链寡核苷酸通过连接整合。所得文库不包含自我互补的发夹形成区域，没有异源双链体，并且可以在Ion Torrent平台上以与基于连接的方案创建的文库相同的效率进行分析。
