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Impact of Polymerase Fidelity on Background Error Rates in Next-Generation Sequencing with Unique Molecular Identifiers/Barcodes.

Journal article
Authors Stefan Filges
Emiko Yamada
Anders Ståhlberg
Tony E Godfrey
Published in Scientific reports
Volume 9
Issue 1
Pages 3503
ISSN 2045-2322
Publication year 2019
Published at Sahlgrenska Cancer Center
Department of Laboratory Medicine
Wallenberg Centre for Molecular and Translational Medicine
Pages 3503
Language en
Subject categories Cancer and Oncology, Other Basic Medicine, Other Medical Biotechnology


Liquid biopsy and detection of tumor-associated mutations in cell-free circulating DNA often requires the ability to identify single nucleotide variants at allele frequencies below 0.1%. Standard sequencing protocols cannot achieve this level of sensitivity due to background noise from DNA damage and polymerase induced errors. Addition of unique molecular identifiers allows identification and removal of errors responsible for this background noise. Theoretically, high fidelity enzymes will also reduce error rates in barcoded NGS but this has not been thoroughly explored. We evaluated the impact of polymerase fidelity on the magnitude of error reduction at different steps of barcoded NGS library construction. We find that barcoding itself displays largest impact on error reduction, even with low fidelity polymerases. Use of high fidelity polymerases in the barcoding step of library construction further suppresses error in barcoded NGS, and allows detection of variant alleles below 0.1% allele frequency. However, the improvement in error correction is modest and is not directly proportional to polymerase fidelity. Depending on the specific application, other polymerase characteristics such as multiplexing capacity, PCR efficiency, buffer requirements and ability to amplify targets with high GC content may outweigh the relatively small additional decrease in error afforded by ultra-high fidelity polymerases.

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