biostudies-literatureZhao MNorthwest Center for Small Fruits ResearchMinisterio de Ciencia e InnovaciónInner Mongolia Agricultural UniversityNHGRI NIH HHSNational Natural Science Foundation of ChinaNational Institutes of Health36https://www.ebi.ac.uk/biostudies/studies/S-EPMC7990137biostudies-literatureUnknown2An unprecedented number of viruses have been discovered by leveraging advances in high-throughput sequencing. Infectious clone technology is a universal approach that facilitates the study of biology and role in disease of viruses. In recent years homology-based cloning methods such as Gibson assembly have been used to generate virus infectious clones. We detail herein the preparation of home-made cloning materials for Gibson assembly. The home-made materials were used in one-step generation of the infectious cDNA clone of a plant RNA virus into a T-DNA binary vector. The clone was verified by a single Illumina reaction and a de novo read assembly approach that required no primer walking, custom primers or reference sequences. Clone infectivity was finally confirmed by <i>Agrobacterium</i>Phytopathology researchHome-made enzymatic premix and Illumina sequencing allow for one-step Gibson assembly and verification of virus infectious clones.PMC7990137U24 HG006620BIO2016-80572-RHG006620U41 HG006620NDGCC2016-2331860489PID2019-109380RBI00 / AEI / 10.13039/501100011033 (AEI-FEDER)Zhao MTzanetakis IESimon-Mateo CGallo AGarcia JAPasin FGarcia BfalseHome-made enzymatic premix and Illumina sequencing allow for one-step Gibson assembly and verification of virus infectious clones.An unprecedented number of viruses have been discovered by leveraging advances in high-throughput sequencing. Infectious clone technology is a universal approach that facilitates the study of biology and role in disease of viruses. In recent years homology-based cloning methods such as Gibson assembly have been used to generate virus infectious clones. We detail herein the preparation of home-made cloning materials for Gibson assembly. The home-made materials were used in one-step generation of the infectious cDNA clone of a plant RNA virus into a T-DNA binary vector. The clone was verified by a single Illumina reaction and a de novo read assembly approach that required no primer walking, custom primers or reference sequences. Clone infectivity was finally confirmed by <i>Agrobacterium</i>2020-01-01T00:00:00Z20202026-04-07T21:02:12.226Z2022-02-09T08:12:26.632ZS-EPMC79901373376897310.1186/s42483-020-00077-4