Fuchs Jonas, Kleine Johanna, Schemmerer Mathias, Kreibich Julian, Maier Wolfgang, Battur Namuun, Krannich Thomas, Sedaghatjoo Somayyeh, Jaki Lena, Maks Anastasija, Boehm Christina, Wilhelm Carina, Schulze Jessica, Mache Christin, Berger Elischa, Panajotov Jessica, Arnold Lisa, Grüning Björn, Bauswein Markus, Böttcher Sindy, Johne Reimar, Wenzel Jürgen, Hölzer Martin, Panning Marcus
Institute of Virology, Freiburg University Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany.
Institute of Clinical Microbiology and Hygiene, National Consultant Laboratory for HAV and HEV, University Medical Center Regensburg, Regensburg, Germany.
Nat Commun. 2025 May 31;16(1):5067. doi: 10.1038/s41467-025-60175-9.
Time- and cost-saving surveillance of viral pathogens is achieved by tiled sequencing in which a viral genome is amplified in overlapping PCR amplicons and qPCR. However, designing pan-specific primers for viral pathogens with high genomic variability represents a significant challenge. Here, we present a bioinformatics command-line tool, called varVAMP (variable virus amplicons), which addresses this issue. It relies on multiple sequence alignments of highly variable virus sequences and enables degenerate primer design for qPCR or tiled amplicon whole genome sequencing. We demonstrate the utility of varVAMP by designing and evaluating novel pan-specific primer schemes suitable for sequencing the genomes of SARS-CoV-2, Hepatitis E virus, rat Hepatitis E virus, Hepatitis A virus, Borna-disease-virus-1, and Poliovirus using clinical samples. Importantly, we also designed primers on the same input data using the software packages PrimalScheme and Olivar and showed that varVAMP minimizes primer mismatches most efficiently. Finally, we established highly sensitive and specific Poliovirus qPCR assays that could potentially simplify current Poliovirus surveillance. varVAMP is open-source and available through PyPI, UseGalaxy, Bioconda, and https://github.com/jonas-fuchs/varVAMP .
通过平铺测序可实现对病毒病原体的省时且经济的监测，即在平铺测序中，病毒基因组在重叠的PCR扩增子和定量PCR中进行扩增。然而，为基因组变异性高的病毒病原体设计泛特异性引物是一项重大挑战。在此，我们展示了一种名为varVAMP（可变病毒扩增子）的生物信息学命令行工具，它解决了这一问题。它依赖于高度可变病毒序列的多序列比对，并能够为定量PCR或平铺扩增子全基因组测序设计简并引物。我们通过设计和评估适用于使用临床样本对严重急性呼吸综合征冠状病毒2、戊型肝炎病毒、大鼠戊型肝炎病毒、甲型肝炎病毒、博尔纳病病毒1和脊髓灰质炎病毒基因组进行测序的新型泛特异性引物方案，证明了varVAMP的实用性。重要的是，我们还使用软件包PrimalScheme和Olivar在相同的输入数据上设计了引物，并表明varVAMP能最有效地减少引物错配。最后，我们建立了高度灵敏且特异的脊髓灰质炎病毒定量PCR检测方法，这可能会简化当前的脊髓灰质炎病毒监测。varVAMP是开源的，可通过PyPI、UseGalaxy、Bioconda以及https://github.com/jonas-fuchs/varVAMP获取。
