Significant Improvements to PCR Amplification Enables Better Performance for A Multitude of Next Generation Sequencing Applications: Library Amplification With Fast, Processive, Higher-Fidelity and Inhibitor Tolerant PCR Master Mixes in The New Era of NGS

Significant Improvements to PCR Amplification Enables Better Performance for A Multitude of Next Generation Sequencing Applications: Library Amplification With Fast, Processive, Higher-Fidelity and Inhibitor Tolerant PCR Master Mixes in The New Era of NGS

Abstract
Most Next-Generation Sequencing (NGS) workflows utilize PCR amplification at some stage of the library preparation process. Targeted short or long read sequencing methods often start with amplifying target regions, followed by incorporating adapter sequences. On the other hand, whole genome or whole transcriptome sequencing requires amplification across the genome or transcriptome after fragmentation and adapter ligation steps. To improve the performance, speed and accuracy of different NGS methods, a fast, processive, high-fidelity and inhibitor-tolerant PCR master mix is required.
Over the last two decades, there has been little advancement in the development of PCR enzymes utilized to amplify libraries for next generation sequencing workflows. Here we demonstrate a novel, newly engineered enzyme and optimally formulated PCR master mix, repliQa HiFi ToughMix, that improves time to results with amplification speeds up to 1 sec/kb for targets < 1 kb, improved processivity for both short and long amplicons (ability to amplify up to 40 kb targets), high fidelity (90 times higher than WT Taq) and superior inhibitor tolerance capabilities. These features of repliQa HiFi ToughMix are beneficial for a wide range of NGS applications. The ability to amplify target regions of the genome with wide GC-content and composition is a very important feature of the PCR master mix for a number of NGS applications. Libraries amplified with repliQa HiFi ToughMix demonstrate unbiased and uniform coverage across difficult target regions ranging from 15% to 85% GC-content, which closely match the data obtained from PCR free libraries. Lower error rates, improved library yield and improved genome coverage also improved the overall quality of the sequencing data. Amplicon-seq applications for both Illumina and long-read sequencing platforms were also benefited by the processivity and speed of this master mix. Finally, amplification of the target DNA for 16S metagenomics or other applications were greatly benefited by the inhibitor tolerance capabilities of repliQa HiFi ToughMix. Overall, repliQa HiFi ToughMix was found to improve results and processing time in a variety of library amplification applications.