Improved transposon-based library preparation for the Ion Torrent platform

Abstract

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.

Keywords: Ion Proton; Ion Torrent; library construction; next-generation sequencing; transposon.

Figure 1. The MuPlus method overcomes limitations of the original transposon-based MuSeek protocol

(A) The presence of stable hairpins and possible formation of template heteroduplexes are two potential causes of low data output with MuSeek libraries. (B) Schematic for MuPlus library preparation protocol workflow. Enzymatic cleavage of the complementary part on one end of the library prevents formation of hairpins and ensures addition of the second adaptor only to homoduplexes.

Figure 2. Frequency of chimeric sequences present in libraries constructed with the three different approaches

Ligation-based library was prepared using the NEBNext Fast DNA Fragmentation & Library Prep Set for Ion Torrent (New England BioLabs); the MuSeek Library Preparation Kit (Thermo Fisher) and the MuPlus method were used to prepare transposon-based libraries. Numbers above bars indicate frequency of chimeric reads per 1,000,000 total reads. Chimeric reads are defined as described in the text.