# Library Preparation Biases Plant Virome Detection: Poly(A) mRNA Enrichment vs. rRNA Depletion in Pepper and Garlic

## TL;DR

This study shows that using rRNA-depleted RNA sequencing gives a more complete picture of plant viruses compared to poly(A)-selected mRNA sequencing.

## Contribution

The study directly compares two RNA library preparation methods for plant virome detection and highlights their biases.

## Key findings

- rRNA-depleted RNA-seq detected more viruses, longer contigs, and complete multipartite DNA virus genomes.
- mRNA-seq was dominated by highly expressed polyadenylated viruses and missed several viruses detected by total RNA-seq.
- Total RNA-seq also detected low-titer RNA viruses in garlic, possibly representing contamination.

## Abstract

High-throughput RNA sequencing reveals plant viromes, but library preparation methods may bias viral detection. Here, we compared rRNA-depleted total RNA-seq and poly(A)-selected mRNA-seq using field-collected pepper leaves (Anseong and Jincheon) and garlic cloves (Hoengseong) from Korean commercial fields. rRNA-depleted total RNA-seq consistently recovered more viruses, longer contigs, and complete multipartite DNA virus genomes (e.g., milk vetch dwarf virus components, tomato spotted wilt virus segments), while mRNA-seq was dominated by highly expressed polyadenylated viruses like broad bean wilt virus 2. In Jincheon pepper, mRNA-seq missed hot pepper endornavirus, pepper cryptic virus 2, and multiple milk vetch dwarf virus segments revealed by total RNA-seq. Garlic libraries showed similar patterns, with total RNA-seq additionally detecting low-titer RNA viruses likely representing contamination. rRNA-depleted total RNA-seq provides a more complete, less biased view of plant viromes and is recommended for comprehensive virus discovery and genome reconstruction, while mRNA-seq remains useful for polyadenylated virus quantification and host gene expression analysis alongside virome profiling.