2.1. High-throughput sequence processing and HAMR analysis

High-throughput sequencing sRNA libraries were retrieved from public datasets (Supplementary Table 1). The resulting sequences were trimmed using Trim Galore (Krueger et al., 2021), filtered on length and quality, and concatenated to produce a single sRNA library that contained multiple sRNA libraries using Galaxy (Afgan et al., 2018). These concatenated fastq files were matched to the corresponding virus or viroid genomes using RNA STAR (Dobin et al., 2013) with the following parameters: outFilterType 1, outFilterMultimapScoreRange 1, outFilterMultimapNmax 10,000, outFilterMismatchNmax 10, outFilterMismatchNoverLmax 0.2, outFilterMismatchNoverReadLmax 1, outFilterScoreMin 0, outFilterScoreMinOverLread 0.66, outFilterMatchNmin 0.

The resulting bam file was used as input in HAMR with the following parameters: with min_read_qual 30, min_read_cov 10, seq_error_rate 0.005, hypothesis H4, max_p 0.05, max_fdr 0.05, refpercent 0.05. Viruses and viroids indicated with * in Fig. 2 used low astringent prediction values in HAMR which were the following: min_read_qual 30, min_read_cov 5, seq_error_rate 0.001, hypothesis H4, max_p 0.05, max_fdr 0.05, refpercent 0.05. For visualization of sRNA mapping (Supplemental Fig. 3), sRNA datasets were aligned to the respective pathogen genomes using bowtie (Langmead et al., 2009) allowing 2 mismatches (v2). The genomes of viruses and viroids used in this study is indicated in Supplementary Table 3).

Characterization of HAMR-predicted modifications in viral and viroid genomes. A. Identification of RNA modifications in viral and viroid pathogens and Arabidopsis mRNAs. B. Total number of modifications identified in viral and viroid pathogens and Arabidopsis mRNAs. C. Total number of modifications identified in viral and viroid pathogens and Arabidopsis mRNAs normalized to genome size. *=significantly enriched, #=significantly depleted. Viruses and viroids indicated with * used low astringent prediction values in HAMR as indicated in the Materials and Methods section.