Fig. 1 | Nature Communications

Fig. 1

From: Direct RNA sequencing on nanopore arrays redefines the transcriptional complexity of a viral pathogen

Fig. 1

Direct RNA sequencing using nanopore arrays is highly reproducible. a Summary metrics for five separate direct RNA-seq runs using normal human dermal fibroblasts (NHDFs) infected with HSV-1 strain Patton GFP-Us11 or HSV-1 strain F vhs null (Δvhs) for either 6 or 18 h. NHDF 18hpi (i) and (ii) represent biological replicates, with an additional technical replicate, NHDF 18hpi (iii), performed on a separate minION device. Calibration strand reads originate from the spiked human enolase 2 (ENO2) mRNA. Pass and fail reads were classified as such by the albacore basecaller. Only reads passing QC (“pass”) were retained for downstream analyses and these were classified by mapping against the HSV-1 genome and H. sapiens transcriptome. Only a small proportion of reads could not be mapped. b The spiking of ENO2 mRNA allows assessment of RNA degradation during library preparation. Here, mRNA degradation is represented by the fraction of ENO2 covered by individual reads and indicates only minimal 5′ degradation during library preparation. c Overview of the nanopore RNA-sequencing methodology. A poly(T) adapter is ligated to poly(A) tails and used to prime first-strand synthesis of cDNA which stabilizes the RNA strand. The poly(T) adapter also allows ligation of the motor protein required to guide the RNA strand through a nanopore

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