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

Abstract

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.

Keywords: Genomics; HPV; Intra-host variation; Library preparation; NGS; SARS-CoV-2; Viral integration; Virology.

Fig. 1

Overview of the TaME-seq2 workflow. The workflow is divided into three steps, primer design shown separately for SARS-CoV-2 and HPV51/52/59, lab workflow consisting of tagmentation step, touch-down PCR and Illumina sequencing, and bioinformatics analysis represented as a flowchart

Fig. 2

Sequencing coverage in representative HPV51, 52, 59, and SARS-CoV-2 positive samples. The coverage plots of A HPV51, B HPV52, C HPV59, and D SARS-CoV-2 aligned to the respective target HPV and SARS-CoV-2 genomes

Fig. 3

Mean number of variants per gene normalized by gene length presented as heatmap. A HPV51, HPV52, and HPV59 positives samples B SARS-CoV-2 positive samples

Fig. 4

Mutational signatures in HPV51, HPV52, HPV59, and SARS-CoV-2 positive clinical samples. The mean proportion of 96 trinucleotide context types is shown below the plots across the different substitution categories. Error bars represent the standard error of the mean