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Anal Biochem. 2015 May 15;477:38-40. doi: 10.1016/j.ab.2014.12.001. Epub 2014 Dec 13.

Increased transcriptome sequencing efficiency with modified Mint-2 digestion-ligation protocol.

Author information

1
Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland. Electronic address: juhana.kammonen@helsinki.fi.
2
Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland.
3
Department of Biosciences, Faculty of Biological and Environmental Sciences, University of Helsinki, 00014 Helsinki, Finland.

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.

KEYWORDS:

Biotin purification; Digestion–ligation; Next-generation sequencing; Sequence-specific miscall

PMID:
25513723
DOI:
10.1016/j.ab.2014.12.001
[Indexed for MEDLINE]

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