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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ascomycota / genetics*
  • Cloning, Molecular
  • Gene Expression Profiling / methods*
  • Gene Library
  • RNA-Directed DNA Polymerase / metabolism*
  • Sequence Analysis / methods*

Substances

  • RNA-Directed DNA Polymerase