Minion mkib

Direct RNA-sequencing libraries were generated from the isolated poly(A) RNA, spiked with 0.25 µl of a synthetic Enolase 2 (ENO2)-derived calibration strand (a 1.3 kb synthetic poly(A)+ RNA, Oxford Nanopore Technologies Ltd.) and sequenced on one of two MinION MkIb with R9.4 flow cells (Oxford Nanopore Technologies Ltd.) and an 18-h runtime. All protocol steps are described in Garalde et al.^{22 (link)}. Following sequencing, basecalling was performed using Albacore 1.2.1 [-f FLO-MIN106 -k SQK-RNA001 -r -n 0 -o fastq, fast5]. Only reads present in the “pass” folder were used in subsequent analyses.

Direct RNA sequencing libraries were generated from 117 to 153 ng of poly(A) RNA, isolated using Dynabeads mRNA purification kit. Isolated poly(A) RNA was subsequently spiked with 0.5 µl of a synthetic Enolase 2 (ENO2) calibration RNA (Oxford Nanopore Technologies Ltd.) and sequenced on a MinION MkIb with R9 flow cells (Oxford Nanopore Technologies Ltd.) for 40 h, as previously described^{14 (link)}. cDNA sequencing libraries were generated from 1 ng of poly(A) RNA, isolated using Dynabeads mRNA purification kit using cDNA-PCR Sequencing kit (SQK-PCS109) (Oxford Nanopore Technologies Ltd.) and sequenced on a MinION MkIb with R9 flow cells for 40 h using the command software MinKNOW v3.1.9 (Oxford Nanopore Technologies). Following basecalling with Guppy v3.2.2, only the reads passing filter were used. Error-correction was performed using proovread as described previously^{14 (link)}. Nanopore read data were aligned to the VZV strain dumas genome (X04370.1) using MiniMap2 v2.15^{41 (link)} and parsed using SAMtools v1.9^{42 (link)} and BEDTools v2.27.1^{43 (link)} before visualizing using the Bioconductor v3.11 and Gviz v1.32.