Abstract
BackgroundGenome variations in human papillomaviruses (HPVs) are common and have been widely investigated in the past two decades. HPV genotyping depends on the finding of the viral genome variations in the L1 ORF. Other parts of the viral genome variations have also been implicated as a possible genetic factor in viral pathogenesis and/or oncogenicity.ResultsIn this study, the HPV58 genome in cervical lesions was completely sequenced both by rolling-circle amplification of total cell DNA and deep sequencing (RCA-seq) and by long PCR template cloning and sequencing. By comparison of three HPV58 genome sequences decoded from three clinical samples to reference HPV-58, we demonstrated that RCA-seq is much more accurate than long-PCR template cloning and sequencing in decoding HPV58 genome. Three HPV58 genomes decoded by RCA-seq displayed a total of 52 nucleotide substitutions from reference HPV58, which could be verified by long PCR template cloning and sequencing. However, the long PCR template cloning and sequencing led to additional nucleotide substitutions, insertions, and deletions from an authentic HPV58 genome in a clinical sample, which vary from one cloned sequence to another. Because the inherited error-prone nature of Tgo DNA polymerase used in preparation of the long PCR templates of HPV58 genome from the clinical samples, the measurable error rate in incorporation of nucleotide into an elongating DNA template was about 0.149% ±0.038% in our studies.ConclusionsSince PCR template cloning and sequencing is widely used in identification of single nucleotide polymorphism (SNP), our data indicate that a serious caution should be taken in finding of true SNPs in various genetic studies.
Highlights
Human papillomaviruses (HPVs) are a group of more than 200 genotypes of small DNA tumor viruses and can be grouped clinically as high-risk types, which are frequently associated with invasive cervical cancer, and low-risk types, which are found mainly in genital warts
Next-generation sequencing (NGS) techniques are emerging as a promising application for human papillomaviruses (HPVs) genotyping and identification of rare single nucleotide polymorphism (SNP) [8,27,28,29,30]
We found that Rolling-circle amplification (RCA) enriched HPV58 genome copy number in the sample 13 from 1154 copies to 7317908 copies while displaying no enrichment activity to host
Summary
Human papillomaviruses (HPVs) are a group of more than 200 genotypes of small DNA tumor viruses (http://pave.niaid.nih.gov/#home) and can be grouped clinically as high-risk (oncogenic) types, which are frequently associated with invasive cervical cancer, and low-risk (non-oncogenic) types, which are found mainly in genital warts. The reported variations have been found in the L1 ORF and the LCR region, and in other parts of the viral genome and were identified mostly, if not all, by PCR amplicon sequencing. It is always questionable whether an authentic variation (s) does exist in the reported HPV genotype because of the use of error-prone Taq polymerase in PCR amplification, many attempts have been made to correlate such genome variations to HPV pathogenesis and viral carcinogenicity [23,24,25,26]. Other parts of the viral genome variations have been implicated as a possible genetic factor in viral pathogenesis and/or oncogenicity
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