Long-Read Sequencing of HSV-1 cDNA

The 1D Strand switching cDNA by ligation protocol (Version: SSE_9011_v108_revS_18Oct2016) from the ONT was used for sequencing HSV-1 cDNAs on the MinION platform. The ONT Ligation Sequencing Kit 1D (SQK-LSK108) was applied for the library preparation using the recommended oligo(dT) primers, or custom-made random oligonucleotides, as well as the SuperScipt IV enzyme for the RTs. The cDNA samples were subjected to PCR reactions with KAPA HiFi DNA Polymerase (Kapa Biosystems) and Ligation Sequencing Kit Primer Mix (part of the 1D Kit). The NEBNext End repair/dA-tailing Module (New England Biolabs) was used for the end repair, whereas the NEB Blunt/TA Ligase Master Mix (New England Biolabs) was utilized for the adapter ligation. The enzymatic steps (e.g.: RT, PCR, and ligation) were carried out in a Veriti Cycler (Applied Biosystems) according to the 1D protocol (Moldován et al., 2018b (link); Tombácz et al., 2018b (link)). The Agencourt AMPureXP system (Beckman Coulter) was used for the purification of samples after each enzymatic reaction. The quantity of the libraries was checked using the Qubit Fluorometer 2.0 and the Qubit (ds)DNAHS Assay Kit (Life Technologies). The samples were run on R9.4 SpotON Flow Cells from ONT.