TaME-seq2: tagmentation-assisted multiplex PCR enrichment sequencing for viral genomic profiling

Background Previously developed TaME-seq method for deep sequencing of HPV, allowed simultaneous identification of the human papillomavirus (HPV) DNA consensus sequence, low-frequency variable sites, and chromosomal integration events. The method has been successfully validated and applied to the st...

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Vydané v:	Virology journal Ročník 20; číslo 1; s. 44
Hlavní autori:	Hesselberg Løvestad, Alexander, Stosic, Milan S., Costanzi, Jean-Marc, Christiansen, Irene Kraus, Aamot, Hege Vangstein, Ambur, Ole Herman, Rounge, Trine B.
Médium:	Journal Article
Jazyk:	English
Vydavateľské údaje:	London BioMed Central 08.03.2023 BioMed Central Ltd Springer Nature B.V BMC
Predmet:	Analysis Bioinformatics Biomedical and Life Sciences Biomedicine carcinogenicity Cervical cancer consensus sequence Conserved sequence COVID-19 COVID-19 Testing DNA DNA sequencing DNA viruses DNA, Viral - genetics Genes Genomes Genomics High-Throughput Nucleotide Sequencing - methods HPV Human papillomavirus Humans Infections Intra-host variation Laboratories Library preparation Methodology Multiplex Polymerase Chain Reaction - methods NGS Nucleotide sequence Nucleotide sequencing Nucleotides Papillomaviridae Papillomaviridae - genetics Papillomavirus Infections Polymerase chain reaction Reproducibility Reproducibility of Results RNA RNA sequencing RNA viruses SARS-CoV-2 SARS-CoV-2 - genetics sequence deletion Severe acute respiratory syndrome coronavirus 2 Viral genetics viral genome Viral infections Virology Norway SARS-CoV-2 Viral integration Genomics Library preparation NGS Intra-host variation HPV Virology
ISSN:	1743-422X, 1743-422X
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Popis
Shrnutí:	Background Previously developed TaME-seq method for deep sequencing of HPV, allowed simultaneous identification of the human papillomavirus (HPV) DNA consensus sequence, low-frequency variable sites, and chromosomal integration events. The method has been successfully validated and applied to the study of five carcinogenic high-risk (HR) HPV types (HPV16, 18, 31, 33, and 45). Here, we present TaME-seq2 with an updated laboratory workflow and bioinformatics pipeline. The HR-HPV type repertoire was expanded with HPV51, 52, and 59. As a proof-of-concept, TaME-seq2 was applied on SARS-CoV-2 positive samples showing the method’s flexibility to a broader range of viruses, both DNA and RNA. Results Compared to TaME-seq version 1, the bioinformatics pipeline of TaME-seq2 is approximately 40× faster. In total, 23 HPV-positive samples and seven SARS-CoV-2 clinical samples passed the threshold of 300× mean depth and were submitted to further analysis. The mean number of variable sites per 1 kb was ~ 1.5× higher in SARS-CoV-2 than in HPV-positive samples. Reproducibility and repeatability of the method were tested on a subset of samples. A viral integration breakpoint followed by a partial genomic deletion was found in within-run replicates of HPV59-positive sample. Identified viral consensus sequence in two separate runs was > 99.9% identical between replicates, differing by a couple of nucleotides identified in only one of the replicates. Conversely, the number of identical minor nucleotide variants (MNVs) differed greatly between replicates, probably caused by PCR-introduced bias. The total number of detected MNVs, calculated gene variability and mutational signature analysis, were unaffected by the sequencing run. Conclusion TaME-seq2 proved well suited for consensus sequence identification, and the detection of low-frequency viral genome variation and viral-chromosomal integrations. The repertoire of TaME-seq2 now encompasses seven HR-HPV types. Our goal is to further include all HR-HPV types in the TaME-seq2 repertoire. Moreover, with a minor modification of previously developed primers, the same method was successfully applied for the analysis of SARS-CoV-2 positive samples, implying the ease of adapting TaME-seq2 to other viruses.
Bibliografia:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23
ISSN:	1743-422X 1743-422X
DOI:	10.1186/s12985-023-02002-5