title: Increased transcriptome sequencing efficiency with modified Mint-2 digestion-ligation protocol.
abstract: The standard digestion-ligation cloning method enables synthesis of large amounts of complementary DNA (cDNA) from a model organism facilitating study of the transcriptome. Here, we used cDNA amplification of the dimorphic yeast Taphrina betulina as an example of how a library construction protocol can significantly increase sequencing throughput. Two modification steps were introduced to the Evrogen standard Mint-2 protocol to improve its suitability for next-generation sequencing projects. We performed two partial Illumina MiSeq sequencing runs with the modified protocol: one with and one without biotin-purified primers. The results demonstrated that biotinylated libraries increased both accuracy and throughput of the modified protocol. Moreover, our sequencing results indicate that a sequence-specific miscall may affect the output of Illumina's MiSeq platform.
authors.affiliation: Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland. Electronic address: juhana.kammonen@helsinki.fi.
authors.forename: Juhana I
authors.initials: JI
authors.lastname: Kammonen
authors.affiliation: Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland.
authors.forename: Olli-Pekka
authors.initials: OP
authors.lastname: Smolander
authors.affiliation: Department of Biosciences, Faculty of Biological and Environmental Sciences, University of Helsinki, 00014 Helsinki, Finland.
authors.forename: Timo
authors.initials: T
authors.lastname: Sipilä
authors.affiliation: Department of Biosciences, Faculty of Biological and Environmental Sciences, University of Helsinki, 00014 Helsinki, Finland.
authors.forename: Kirk
authors.initials: K
authors.lastname: Overmyer
authors.affiliation: Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland.
authors.forename: Petri
authors.initials: P
authors.lastname: Auvinen
authors.affiliation: Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland.
authors.forename: Lars
authors.initials: L
authors.lastname: Paulin
mesh_terms: D001203:Ascomycota / Q000235:genetics*; D003001:Cloning, Molecular; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D017421:Sequence Analysis / Q000379:methods*
chemical_list: D012194:RNA-Directed DNA Polymerase
keywords: Biotin purification; Digestion–ligation; Next-generation sequencing; Sequence-specific miscall