ena-STUDY-Friedrich-Loeffler-Institute-13-12-2018-08:21:32:262-146
Custom Y-adapters for improved library preparation for Ion Torrent sequencing
Library preparation is a crucial step in preparing samples for next-generation sequencing, at least partly determining the success of a sequencing project. A key issue of library preparation is the amount of input DNA, which is often limited and may hence result in low output of the final library. To compensate this library amplification is often performed as an additional step of library preparation. However, this introduces biases and reduces the complexity of the library. One possibility to improve the efficiency of library preparation is the use of Y-shaped adapters combined with T-A-ligation. Here, we designed and validated custom Y-adapters compatible with Ion Torrent platforms. According to our analyses, the critical determinants of the adapter performance are the length of the double stranded stem (optimum 12 bp) and the corresponding melting temperature (optimum range 45°C – 55°C), as well as the incorporation of at least one phosphorothioate bond at the ends of the oligonucleotides to prevent from adapter degradation. Extensive testing with optimised custom Y-adapters showed that the library yield is higher compared with commercial Ion Torrent standard adapters while the sequencing performance, library complexity, read length and quality equals that of standard adapters.
Library preparation is a crucial step in preparing samples for next-generation sequencing, at least partly determining the success of a sequencing project. A key issue of library preparation is the amount of input DNA, which is often limited and may hence result in low output of the final library. To compensate this library amplification is often performed as an additional step of library preparation. However, this introduces biases and reduces the complexity of the library. One possibility to improve the efficiency of library preparation is the use of Y-shaped adapters combined with T-A-ligation. Here, we designed and validated custom Y-adapters compatible with Ion Torrent platforms. According to our analyses, the critical determinants of the adapter performance are the length of the double stranded stem (optimum 12 bp) and the corresponding melting temperature (optimum range 45°C – 55°C), as well as the incorporation of at least one phosphorothioate bond at the ends of the oligonucleotides to prevent from adapter degradation. Extensive testing with optimised custom Y-adapters showed that the library yield is higher compared with commercial Ion Torrent standard adapters while the sequencing performance, library complexity, read length and quality equals that of standard adapters.