Vol 4: Long-range PCR in next-generation sequencing: comparison of six enzymes and evaluation on the MiSeq sequencer.Report as inadecuate



 Vol 4: Long-range PCR in next-generation sequencing: comparison of six enzymes and evaluation on the MiSeq sequencer.


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This article is from Scientific Reports, volume 4.AbstractLong-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.



Author: Jia, Haiying; Guo, Yunfei; Zhao, Weiwei; Wang, Kai

Source: https://archive.org/







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