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Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Published on: June 3, 2019

Quality control and statistical evaluation of combinatorial DNA libraries using nanopore sequencing.

Cédric Lood^1,2, Hans Gerstmans^1,3,4, Yves Briers³

¹Department of Biosystems, Laboratory of Gene Technology, KU Leuven, Leuven, Belgium.

|October 1, 2020

Summary

This study introduces a new method using long-read sequencing to fully evaluate DNA building block libraries for protein engineering. This approach ensures library diversity and quality before downstream applications.

Keywords:

DNA assembly GoldenBraid assembly GoldenGate assembly VersaTile shuffling combinatorial DNA library constructs validation nanopore sequencing quality control synthetic biology

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Area of Science:

Molecular Biology
Synthetic Biology
Bioinformatics

Background:

Modular DNA libraries are crucial for protein engineering and synthetic biology.
Current validation methods (Sanger sequencing) are limited, analyzing only a small subset of clones.
Ad hoc validation lacks comprehensiveness for large combinatorial libraries.

Purpose of the Study:

To implement a systematic procedure for comprehensive evaluation of DNA building block libraries.
To validate library composition and diversity immediately after in vitro creation.
To improve the reliability of protein engineering and synthetic biology workflows.

Main Methods:

Utilized long-read sequencing technology (Nanopore sequencing).
Employed straightforward bioinformatics tools for data analysis.
Applied exploratory statistics to assess library diversity and composition.

Main Results:

Developed a systematic procedure for comprehensive library evaluation.
Enabled tabulation of building block composition and synteny from sequencing reads.
Provided statistical assessment of library diversity.

Conclusions:

The new method offers a comprehensive and systematic approach to validate combinatorial DNA libraries.
This validation ensures library quality and diversity, crucial for downstream applications.
The procedure enhances the reliability of protein engineering and synthetic biology endeavors.