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Updated: Apr 19, 2026

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

Published on: November 18, 2014

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

Juhana I Kammonen¹, Olli-Pekka Smolander¹, Timo Sipilä²

¹Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland.

Analytical Biochemistry

|December 17, 2014

Summary

This summary is machine-generated.

We optimized a complementary DNA (cDNA) library construction protocol for yeast, enhancing sequencing throughput and accuracy. Biotinylated primers significantly improved results on the Illumina MiSeq platform, suggesting potential sequence-specific miscalls.

Keywords:

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

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Last Updated: Apr 19, 2026

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

Published on: November 18, 2014

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

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Published on: September 16, 2019

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

Published on: May 5, 2018

Area of Science:

Molecular Biology
Genomics
Next-Generation Sequencing

Background:

Complementary DNA (cDNA) synthesis is crucial for transcriptome studies.
Standard cloning methods can be limiting for high-throughput sequencing.
Optimizing library construction protocols is essential for efficient next-generation sequencing (NGS).

Purpose of the Study:

To modify and enhance a cDNA library construction protocol for improved sequencing throughput.
To evaluate the impact of biotinylated primers on sequencing accuracy and efficiency.
To investigate potential sequence-specific miscalls on the Illumina MiSeq platform.

Main Methods:

cDNA amplification from the dimorphic yeast Taphrina betulina.
Modification of the Evrogen Mint-2 protocol for NGS suitability.
Illumina MiSeq sequencing of libraries prepared with and without biotinylated primers.

Main Results:

The modified protocol significantly increased sequencing throughput.
Biotinylated libraries demonstrated higher accuracy and throughput compared to non-biotinylated libraries.
A potential sequence-specific miscall affecting Illumina MiSeq output was identified.

Conclusions:

The modified cDNA library construction protocol enhances NGS efficiency.
Biotinylated primers are beneficial for improving accuracy and throughput in yeast transcriptome sequencing.
Further investigation into sequence-specific miscalls on Illumina MiSeq is warranted.