Nanopore sequencing for Viral pUG RNAs

N2 or YY1449 animals were infected with Orsay virus for 3-4 days. Animals were collected and RNA-extracted with TRIzol and chloroform. The total RNA was depleted of rRNA using RNAse-H based method for depletion. Spike-in gfp pUG RNAs were added to normalize the libraries. Reverse transcription was performed with poly(AC) RT primers. The newly synthesized cDNA was amplified with LongAmp Taq Polymerase for 8 cycles, then depleted of yrn-1 and RT artifacts using the CRISPR/Cas9 with the DASH protocol. The DASH-treated cDNAs were then PCR-barcoded with the Oxford Nanopore PCR-barcoding protocol before proceeding to sequencing. Briefly, the pooled library was loaded into MinION r10 flow cells on a MinION Mk1C machine (Oxford Nanopore). Sequencing was set to run for at least 24 hours, barcoding was turned on with no trimming, and quality score filter of 9. Guppy high accuracy base calling model was used. Fastq files that passed the quality score filter were in the fastq_pass folder. The fastq files were merged using cat *.fastq. Next, pychopper was used to identify and orient full-length transcripts. The PCS111_primers.fas was modified as following: >VNP TTGCCTGTCGCTCTATCTTC >SSP TCTGTTGGTGCTGATATTGCTTT Pychopper was used with the following settings: -m edlib -p -k PCS111. The output fastq files were then merged and filtered for TG repeats using the following: grep -B1 -A2 --no-group-separator -E .GTGTGTGTGT.{1\} Then, the filtered fastq files were then used to map to the transcriptome, spike-in, and the Orsay virus using minimap2 -uf k14. The resulting sam files were converted to bam files that were indexed and sorted using samtools. The libraries were normalized to RPM using the spike-in gfp pUG RNAs to normalize the libraries for any technical variations from the library generation. Sam files were processed using pysam to analyze the soft clipped sequences that correspond to the pUG tails as well as identifying the location of pUG tail sites.