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Increased transcriptome sequencing efficiency with modified Mint-2 digestion–ligation protocol
- Kammonen, Juhana I., Smolander, Olli-Pekka, Sipilä, Timo, Overmyer, Kirk, Auvinen, Petri, Paulin, Lars
- Analytical biochemistry 2015 v.477 pp. 38-40
- Taphrina, complementary DNA, high-throughput nucleotide sequencing, models, protocols, transcriptome, transcriptomics, yeasts
- 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.