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DOI: 10.1101/2022.12.22.22283851

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

A.Hesselberg Lovestad M. Stosic J.-M. Costanzi ...+3 T. B. Rounge
摘要
Background Previously developed TaME-seq method for deep sequencing of HPV, allowed simultaneous identification of the 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 40x faster. In total, 23 HPV-positive samples and seven SARS-CoV-2 clinical samples passed the threshold of 300x mean depth and were submitted to further analysis. The mean number of variable sites per 1000 bp was ~ 1.5x 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.
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