Open AccessImproved transposon-based library preparation for the Ion Torrent platform
Author(s) -
Tatyana Gorbacheva,
Wilber Quispe-Tintaya,
В. Н. Попов,
Jan Vijg,
Alexander Y. Maslov
Publication year - 2015
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/000114277
Subject(s) - ion semiconductor sequencing , computational biology , oligonucleotide , transposable element , transposase , library , genomic library , sequencing by ligation , biology , dna , ligation , dna sequencing , genetics , microbiology and biotechnology , base sequence , gene , genome , 16s ribosomal rna
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.