Department of Pathology and Genetics, Sahlgrenska Cancer Center, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Medicinaregatan 1F, 405 30, Gothenberg, Sweden.
Department of Surgery, Boston University School of Medicine, 700 Albany Street, Boston, MA, 02118, USA.
Sci Rep. 2019 Mar 5;9(1):3503. doi: 10.1038/s41598-019-39762-6.
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.
液体活检和检测游离循环 DNA 中的肿瘤相关突变通常需要能够识别等位基因频率低于 0.1%的单核苷酸变异。由于 DNA 损伤和聚合酶诱导错误引起的背景噪声，标准测序方案无法达到这种灵敏度水平。添加独特的分子标识符允许识别和消除导致这种背景噪声的错误。理论上，高保真酶也将降低 barcoded NGS 中的错误率，但这尚未得到彻底探索。我们评估了聚合酶保真度对条形码 NGS 文库构建不同步骤中错误减少幅度的影响。我们发现，即使使用低保真度聚合酶，条形码本身对错误减少的影响最大。在文库构建的条形码步骤中使用高保真度聚合酶进一步抑制了条形码 NGS 中的错误，并允许检测到等位基因频率低于 0.1%的变异等位基因。然而，错误校正的改善是适度的，并且与聚合酶保真度不成正比。根据具体应用，其他聚合酶特性，如多重性、PCR 效率、缓冲液要求以及扩增具有高 GC 含量的靶标的能力，可能超过超保真聚合酶提供的相对较小的额外错误降低。
