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Volume 10, Issue 4

Identifying the best PCR enzyme for library amplification in NGS Open Access

Abstract

PCR amplification is a necessary step in many next-generation sequencing (NGS) library preparation methods [ 1, 2 ]. Whilst many PCR enzymes are developed to amplify single targets efficiently, accurately and with specificity, few are developed to meet the challenges imposed by NGS PCR, namely unbiased amplification of a wide range of different sizes and GC content. As a result PCR amplification during NGS library prep often results in bias toward GC neutral and smaller fragments. As NGS has matured, optimized NGS library prep kits and polymerase formulations have emerged and in this study we have tested a wide selection of available enzymes for both short-read Illumina library preparation and long fragment amplification ahead of long-read sequencing.

We tested over 20 different hi-fidelity PCR enzymes/NGS amplification mixes on a range of Illumina library templates of varying GC content and composition, and find that both yield and genome coverage uniformity characteristics of the commercially available enzymes varied dramatically. Three enzymes Quantabio RepliQa Hifi Toughmix, Watchmaker Library Amplification Hot Start Master Mix (2X) ‘Equinox’ and Takara Ex Premier were found to give a consistent performance, over all genomes, that mirrored closely that observed for PCR-free datasets. We also test a range of enzymes for long-read sequencing by amplifying size fractionated DNA of average size 21.6 and 13.4 kb, respectively.

The enzymes of choice for short-read (Illumina) library fragment amplification are Quantabio RepliQa Hifi Toughmix, Watchmaker Library Amplification Hot Start Master Mix (2X) ‘Equinox’ and Takara Ex Premier, with RepliQa also being the best performing enzyme from the enzymes tested for long fragment amplification prior to long-read sequencing.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): amplification , barcode , illumina , indexing , next-generation sequencing , PCR and polymerase
Funding
This study was supported by the:
  • Wellcome Trust (Award 098051)
    • Principle Award Recipient: MichaelA Quail
© 2024 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2024-04-05
2024-04-29
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Identifying the best PCR enzyme for library amplification in NGS
M Gen 10, 001228 (2024); https://doi.org/10.1099/mgen.0.001228
/content/journal/mgen/10.1099/mgen.0.001228
/content/journal/mgen/10.1099/mgen.0.001228
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