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. 2021 Dec 11;25(1):103613.
doi: 10.1016/j.isci.2021.103613. eCollection 2022 Jan 21.

Translating genomic exploration of the family Polyomaviridae into confident human polyomavirus detection

Sergio Kamminga  1   2 , Igor A Sidorov  1 , Michaël Tadesse  1 , Els van der Meijden  1 , Caroline de Brouwer  1 , Hans L Zaaijer  2 , Mariet C W Feltkamp  1 , Alexander E Gorbalenya  1   3
Affiliations

Translating genomic exploration of the family Polyomaviridae into confident human polyomavirus detection

Sergio Kamminga et al. iScience. .

Abstract

The Polyomaviridae is a family of ubiquitous dsDNA viruses that establish persistent infection early in life. Screening for human polyomaviruses (HPyVs), which comprise 14 diverse species, relies upon species-specific qPCRs whose validity may be challenged by accelerating genomic exploration of the virosphere. Using this reasoning, we tested 64 published HPyV qPCR assays in silico against the 1781 PyV genome sequences that were divided in targets and nontargets, based on anticipated species specificity of each qPCR. We identified several cases of problematic qPCR performance that were confirmed in vitro and corrected through using degenerate oligos. Furthermore, our study ranked 8 out of 52 tested BKPyV qPCRs as remaining of consistently high quality in the wake of recent PyV discoveries and showed how sensitivity of most other qPCRs could be rescued by annealing temperature adjustment. This study establishes an efficient framework for ensuring confidence in available HPyV qPCRs in the genomic era.

Keywords: Omics; Virology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Dynamic of accumulation of complete polyomavirus genome sequences Shown is annual dynamic of accumulation of the analyzed complete genome sequences of the family Polyomaviridae in GenBank until October 10th 2019 (1781 genomes), according to HAYGENS (https://veb.lumc.nl/HAYGENS/). Genome sequences are dated according to their GenBank entries, which may deviate from the first date of the public sequence release.
Figure 2
Figure 2
Schematic workflow of in silico PCR testing and example of results visualization Presented are main stages of in silico analysis of a publicly available HPyV qPCR using genome sequences of polyomaviruses (top two panels), as well as an example of results visualization (bottom panel). This pipeline is also applied to analysis of in-house HPyV qPCRs, which provided PCR variables. All calculations are performed for each genome sequence and PCR oligos set and are detailed in the STAR Methods section. Results of in silico evaluation of the qPCR in respect to T-decision ranges and L-decision binary of qPCR oligos annealed to target (blue) and nontarget (red) templates are presented using a Tm map for each pair of qPCR oligos, three in total. Each Tm map is divided into four nonoverlapping orthogonal zones delimited by two internal boundaries set at temperature (T) corresponding to the Ta of the presented qPCR and distinguished by three background colors. Light blue zone: T-ranges favorable for annealing of both oligos to template and facilitating qPCR; light red zone: T-ranges unfavorable for both oligos to facilitate qPCR; two light gray zones: a T-range is unfavorable for one of two oligos to facilitate qPCR. When the coordinates of the calculated qPCR product conforms to the sequence location boundaries delimited by the corresponding L-decision = 1, Tm values of a pair of oligos annealed to the respective sequence are labeled with a circle, otherwise they are a diamond. Two labels may overlap, partly or fully, on the map, and size of circle or diamond label is proportional to the number of labels, when they fully overlap. Position of each label on the map corresponds to Tm of the oligo/template complex for two oligos under which the T-decision is equal to 0.5. Each label occupies middle position in two bars, vertical (bottom-to-top) and horizontal (left-to-right), which delimit T-ranges for the respective oligo/template complexes corresponding to the T-decision [0.95–0.05] ranges (see STAR Methods). The opacity of each label corresponds to its T-decision value within the respective target or nontarget color gradient. Interactive versions of 2D and 3D Tm maps for the data presented in this study are available on the resource website. The user can zoom into any label to learn from a pop-up about Tm, template GenBank ID, and other characteristics of oligo/template complexes. When a label represents several fully overlapping oligo/template complexes, the pop-up informs about the number of sequences involved in the overlap and details characteristics of a single sequence only. Note that an overlap may involve sequences from the same or different groups, namely targets and nontargets. The user may explore three 2D Tm maps of a PCR simultaneously using a 3D Tm map that can facilitate understanding the basis of sensitivity and selectivity.
Figure 3
Figure 3
In silico and in vitro testing and refinement of a TSPyV qPCR (A and B) Results of in silico testing of the in-house TSPyV qPCR under standard Ta for qPCR oligos annealed to 23 target (blue) and 1758 nontarget (red) templates are presented using two oligo/template Tm maps: forward versus reverse primer pair (A), forward primer versus reverse probe pair (B). A Tm shift for oligo/template complex of seven TSPyV genomes, typified by KM007161.1, after including a degenerated base into the forward primer (Table S3) is shown with the bold arrow. Overall design of the Tm maps is explained in the legend of Figure 2. (C) In vitro dilution series of two TSPyV genomes with either full-match (NC_014361) (qPCR efficiency = 97.6%, R2 = 0.996, slope = −3.382) or a mismatch (KM007161.1) to the forward oligo (qPCR efficiency = 97.1%, R2 = 0.995, slope = −3.394). A relative poor recognition of the mismatch genome is evident. (D) The same as in (C) except for using forward primer with a degenerate base. Note similar recognition of the two genomes.
Figure 4
Figure 4
In silico and in vitro testing of a JCPyV qPCR (A–C) Results of in silico evaluation of the in-house JCPyV qPCR under standard Ta for qPCR oligos annealed to 690 target (blue) and 1,091 nontarget (red) templates are presented using three oligo/template Tm maps: forward versus reverse primer oligo pair (A); forward primer versus forward probe oligo pair (B); and reverse primer versus forward probe oligo pair (C). Overall design of the Tm maps is explained in the legend of Figure 2. Fully interactive versions of these maps and a 3D melting temperature map are available on the resource website. (D) The influence of a common mismatch present in 72/690 JCPyV genomes was tested by comparing the performance of the qPCR on the regular control plasmid without mismatch (Mismatch−, efficiency = 98.7%, R2 = 0.998, slope = −3.353) and the plasmid containing the mismatch (Mismatch+, efficiency = 104.4%, R2 = 0.994, slope = −3.222) (D) (e.g. AF015535.1, forward primer annealing region: GTCTCCCCAT[A→G]CCAACATTAGCTT). A small difference in Cq values is seen when the mismatch is present.
Figure 5
Figure 5
In silico and in vitro testing of a BKPyV qPCR (A–C) Results of in silico testing of the in-house BKPyV qPCR under standard Ta for qPCR oligos annealed to 522 target (blue) and 1,259 nontarget (red) sequences are presented using three quadrant oligo/template Tm maps as detailed in legend to Figure 2: forward versus reverse primer oligo pair (A); forward primer versus forward probe oligo pair (B); and reverse primer versus forward probe oligo pair (C). Selected genome sequences discussed in the text are indicated with arrows accompanied by their GenBank numbers. (D) In vitro evaluation of impact of a common single nucleotide mismatch in the probe-to-target annealing region on the qPCR performance against GenBank ID AB211375.1. An increase of about 1 Cq for the target with the mismatch (efficiency = 94%, R2 = 0.993, slope = −3.475) relative to the matching target was observed (efficiency = 94.7%, R2 = 0.999, slope = −3.456).
Figure 6
Figure 6
Sensitivity and selectivity for BKPyV qPCRs under original and adjusted Ta For each published BKPyV qPCR specified at the right, selectivity and sensitivity are depicted schematically with contrasting colors under original and adjusted Ta along with difference between these Ta. Impact of Ta adjustment is shown as increase (SN+, SL+) or decrease (SN−, SL−) of the corresponding original sensitivity and selectivity values (SN and SL). PCRs are listed in the descending order according to the sensitivity under adjusted Ta. Ta difference = (adjusted Ta−original Ta) is shown with gray bars.
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