title: Long-range PCR in next-generation sequencing: comparison of six enzymes and evaluation on the MiSeq sequencer.
abstract: Long-range PCR remains a flexible, fast, efficient and cost-effective choice for sequencing candidate genomic regions in a small number of samples, especially when combined with next-generation sequencing (NGS) platforms. Several long-range DNA polymerases are advertised as being able to amplify up to 15 kb or longer genomic DNA. However, their real-world performance characteristics and their suitability for NGS remain unclear. We evaluated six long-range DNA polymerases (Invitrogen SequalPrep, Invitrogen AccuPrime, TaKaRa PrimeSTAR GXL, TaKaRa LA Taq Hot Start, KAPA Long Range HotStart and QIAGEN LongRange PCR Polymerase) to amplify three amplicons, with sizes of 12.9 kb, 9.7 kb, and 5.8 kb, respectively. Subsequently, we used the PrimeSTAR enzyme to amplify entire BRCA1 (83.2 kb) and BRCA2 (84.2 kb) genes from nine subjects and sequenced them on an Illumina MiSeq sequencer. We found that the TaKaRa PrimeSTAR GXL DNA polymerase can amplify almost all amplicons with different sizes and Tm values under identical PCR conditions. Other enzymes require alteration of PCR conditions to obtain optimal performance. From the MiSeq run, we identified multiple intronic and exonic single-nucleotide variations (SNVs), including one mutation (c.5946delT in BRCA2) in a positive control. Our study provided useful results for sequencing research focused on large genomic regions.
authors.affiliation: 1] The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong 510632, China [2] Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA.
authors.forename: Haiying
authors.initials: H
authors.lastname: Jia
authors.affiliation: 1] Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA [2] Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA.
authors.forename: Yunfei
authors.initials: Y
authors.lastname: Guo
authors.affiliation: Kingmed Diagnostics, Guangzhou, Guangdong 510330, China.
authors.forename: Weiwei
authors.initials: W
authors.lastname: Zhao
authors.affiliation: 1] Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA [2] Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA [3] Department of Psychiatry, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Wang
mesh_terms: D004252:DNA Mutational Analysis / Q000191:economics / Q000295:instrumentation* / Q000379:methods; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D019398:Genes, BRCA1; D024522:Genes, BRCA2; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D017384:Sequence Deletion
chemical_list: D004259:DNA-Directed DNA Polymerase