Quality control of next-generation sequencing library through an integrative digital microfluidic platform

Area of Science:

• Biotechnology
• Analytical Chemistry
• Genomics

Background:

• Next-generation sequencing (NGS) library preparation requires precise quality control (QC) for optimal performance.
• Current QC methods can be sample-intensive, limiting the analysis of precious samples.
• Automated and sensitive QC platforms are needed to streamline NGS workflows.

Purpose of the Study:

• To develop an automated quality control (QC) platform for next-generation sequencing (NGS) library characterization.
• To integrate droplet-based digital microfluidics (DMF) with capillary electrophoresis (CE) for enhanced sample analysis.
• To reduce sample volume requirements while maintaining high sensitivity and accuracy.

Main Methods:

• Integration of a droplet-based digital microfluidic (DMF) system with a capillary-based reagent delivery unit and a quantitative capillary electrophoresis (CE) module.
• Development of an in-plane capillary-DMF interface for electrokinetic injection.
• Utilized a DNA ladder as an internal standard for quantitative detection of double-stranded DNA (dsDNA).

Main Results:

• The developed DMF-CE platform demonstrated sensitive detection of dsDNA in the range of 5-100 pg/μL.
• The platform consumes tenfold less sample volume compared to the Agilent BioAnalyzer QC technique.
• Limited-cycle PCR amplification was shown to bias library size distribution towards larger fragments.

Conclusions:

• The automated DMF-CE platform provides sensitive and accurate QC for NGS libraries, preserving precious samples.
• This microfluidic system is suitable for the DNA quantification needs of platforms like the Illumina Genome Analyzer.
• The platform effectively validates NGS library preparation, revealing biases introduced by PCR amplification.