Identifying the best PCR enzyme for library amplification in NGS

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Polymerase Chain Reaction (PCR) amplification is essential for next-generation sequencing (NGS) library preparation.
• Standard PCR enzymes often introduce bias in fragment size and GC content during NGS library preparation.
• Optimized PCR formulations are needed to address the challenges of unbiased amplification for diverse NGS applications.

Purpose of the Study:

• To evaluate the performance of various high-fidelity PCR enzymes for both short-read (Illumina) and long-read sequencing library preparation.
• To identify PCR enzymes that provide consistent and unbiased amplification across different DNA fragment sizes and GC content.
• To compare enzyme performance against PCR-free datasets for Illumina library preparation.

Main Methods:

• Tested over 20 high-fidelity PCR enzymes and NGS amplification mixes.
• Evaluated enzyme performance on Illumina library templates with varying GC content and composition.
• Assessed enzyme suitability for long-read sequencing by amplifying size-fractionated *S. cerevisiae* DNA fragments (13.4 kb and 21.6 kb).

Main Results:

• Significant variation in yield and genome coverage uniformity was observed among tested PCR enzymes.
• Quantabio RepliQa Hifi Toughmix, Watchmaker Library Amplification Hot Start Master Mix (2X) 'Equinox', and Takara Ex Premier demonstrated consistent performance for Illumina library preparation.
• These three enzymes closely mirrored the results obtained from PCR-free datasets.
• Quantabio RepliQa Hifi Toughmix also showed superior performance for long fragment amplification for long-read sequencing.

Conclusions:

• Quantabio RepliQa Hifi Toughmix, Watchmaker 'Equinox', and Takara Ex Premier are recommended for unbiased short-read Illumina library amplification.
• Quantabio RepliQa Hifi Toughmix is also the top choice for long fragment amplification prior to long-read sequencing.
• Selecting appropriate PCR enzymes is critical for minimizing bias and maximizing data quality in NGS workflows.