EBV B95-8-BAC nanopore direct RNA-seq latency transcriptome
Direct RNA-seq and subsequent analysis of four LCLs established in mixed genetic donor B cells. The virus used was a subclone of the Epstein-Barr virus (EBV; HHV4)  B95-8 strain BAC originally cloned by Delecluse and Hammerschmict (10.1073/pnas.95.14.8245). This clone (pHB9) contains six repeat units in major internal repeat IR1. We have previously shown that the major internal repeat IR1 (Bam W repeat) has one repeat unit that contains a STOP codon in EBNA-LP exon W1 (Ba abdullah et al 2017 - 10.1128/JVI.00920-17). We have generated an additional EBV BAC clone (pWTw) that contains a fully homogenous IR1 lacking this STOP codon (Szymula et al 2018 - 10.1371/journal.ppat.1006890). Here we have generated an additional clone in which distinct translationally silent two nucleotide modifications (barcodes) have been introduced into exon W2 of the first and last IR1 repeat units (pHB9-W4-MR11+x.y) and used this to generate two 293 cell clones for packaging this as a viral genome (clones AJ2 and MJ2). These four viruses were used to establish LCLs, and the RNA from these LCLs was extracted and analysed by nanopore direct RNA-seq (version 9 MinION flow cells). The aims of the project were: 1.  to characterise the nature of transcripts spanning IR1, to define the origin of different sized EBNA-LP proteins. 2. to identify which repeat unit contains the stop codon in B95-8 ebb strain.  3. To improve characterisation and annotation of the EBV-BAC transcriptome 4. characterising relationships/co-ordination between alternative splicing events, promoter usage, and poyadenylation site usage.    Aligning of reads to the EBV genome failed to correctly align the 5' ends of reads appropriately to the W0 geneome. We developed a script to re-align the otherwise soft-clipped w0 exon part fo the read. The data in this archive are:       1. Fastq files for all reads (host and EBV) that pass minimum quality thresholds; Bam files and associated index files, corrected to align Exon W0 (both under experiment ERX13531966-69).        2. Raw Nanopore Fast5 file for the subset of reads that align to the EBV genome (under experiment ERX13531970-73).