Mosquera-Yuqui Francisco, Ramos-Lopez Daniel, Hu Xiaojun, Yang Yu, Mendoza Joshua L, Asare Emmanuel, Habiger Joshua, Hurtado-Gonzales Oscar P, Espindola Andres S
Institute for Biosecurity and Microbial Forensics (IBMF), Oklahoma State University, Stillwater, OK, USA.
Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, USA.
Sci Rep. 2025 Jan 11;15(1):1657. doi: 10.1038/s41598-025-86065-0.
Exclusion is a keystone of integrated pest management to prevent the introduction of pathogens. U.S. plant quarantine programs employ PCR and high-throughput sequencing (HTS) to test imported plants for viruses and viroids of concern. Achieving a low limit of detection in any HTS protocol could be challenging. Following a template-switching cDNA amplification protocol, seven cDNA synthesis treatments were used to test simultaneously the relative abundance and coverage of the three most commonly latent RNA viruses found in apples: apple chlorotic leaf spot virus, apple stem grooving virus, and apple stem pitting virus, as well as the viroid apple hammerhead viroid. Amplified double-stranded cDNAs were subjected to library preparation using Nanopore SQK-DCS109 and Illumina Nextera XT, and sequenced with MinION and NextSeq2000, respectively. Treatments with oligo d(T)23-VN or its combination with random hexamers yielded the highest relative reads for viruses, while treatments containing the reverse primer pool produced more relative reads for AHVd. These treatments and random hexamers also generated the highest genome coverages, which were typically similar in both HTS workflows. However, relative abundances of viruses determined with SQK-DCS109 were up to 2.22-fold higher compared to Nextera XT. In contrast, Nextera XT yielded viroid reads 3.30-fold higher than SQK-DCS109. A framework of considerations for expanding this sensitive approach to other targets and crops is discussed.
排除是综合虫害管理中防止病原体引入的关键要素。美国植物检疫计划采用聚合酶链式反应（PCR）和高通量测序（HTS）来检测进口植物是否携带相关病毒和类病毒。在任何高通量测序方案中实现低检测限都可能具有挑战性。遵循模板转换cDNA扩增方案，使用七种cDNA合成处理方法同时检测苹果中三种最常见的潜伏RNA病毒的相对丰度和覆盖率：苹果褪绿叶斑病毒、苹果茎沟病毒和苹果茎痘病毒，以及类病毒苹果锤头状类病毒。扩增的双链cDNA使用Nanopore SQK-DCS109和Illumina Nextera XT进行文库制备，并分别用MinION和NextSeq2000进行测序。用寡聚d(T)23-VN或其与随机六聚体的组合处理产生了最高的病毒相对读数, 而含有反向引物池的处理产生了更多的苹果锤头状类病毒相对读数。这些处理和随机六聚体也产生了最高的基因组覆盖率，这在两种高通量测序工作流程中通常相似。然而，与Nextera XT相比，用SQK-DCS109测定的病毒相对丰度高出2.22倍。相比之下，Nextera XT产生的类病毒读数比SQK-DCS109高3.30倍。本文讨论了将这种灵敏方法扩展到其他目标和作物的考虑框架。
