A common challenge in pathogen discovery by deep sequencing approaches is to recognize viral or subviral pathogens in samples of diseased tissue that share no significant homology with a known pathogen. Here we report a homology-independent approach for discovering viroids, a distinct class of free circular RNA subviral pathogens that encode no protein and are known to infect plants only. Our approach involves analyzing the sequences of the total small RNAs of the infected plants obtained by deep sequencing with a unique computational algorithm, progressive filtering of overlapping small RNAs (PFOR). Viroid infection triggers production of viroid-derived overlapping siRNAs that cover the entire genome with high densities. PFOR retains viroid-specific siRNAs for genome assembly by progressively eliminating nonoverlapping small RNAs and those that overlap but cannot be assembled into a direct repeat RNA, which is synthesized from circular or multimeric repeated-sequence templates during viroid replication. We show that viroids from the two known families are readily identified and their fulllength sequences assembled by PFOR from small RNAs sequenced from infected plants. PFOR analysis of a grapevine library further identified a viroid-like circular RNA 375 nt long that shared no significant sequence homology with known molecules and encoded active hammerhead ribozymes in RNAs of both plus and minus polarities, which presumably self-cleave to release monomer from multimeric replicative intermediates. A potential application of the homology-independent approach for viroid discovery in plant and animal species where RNA replication triggers the biogenesis of siRNAs is discussed. T he term "viroid" was first introduced in 1971 to describe a novel class of free RNA nonviral pathogens found in plants (1-3). Viroids are single-stranded circular RNA molecules of 246-401 nt in length and do not encode any protein. Viroid RNAs display extensive intramolecular base pairing to give rod-like or quasirod-like conformations. Replication of viroids occurs via a rolling circle mechanism by host RNA polymerases to yield head-to-tail multiple-repeat replicative intermediates. Viroids identified to date infect only plants and belong to one of the two families. Viroids in the Pospiviroidae such as potato spindle tuber viroid (PSTVd) share a five-domain model including a central conserved region (CCR) and may all replicate in the nucleus. By contrast, viroids in the Avsunviroidae such as peach latent mosaic viroid (PLMVd) lack a CCR, encode a hammerhead ribozyme in both the positive and the negative strands that self-cleaves to release monomer from the multimeric intermediates, and may all replicate in the chloroplasts (1-3).Identification of new viroids requires purification and enrichment of the naked viroid RNA by 2D gel electrophoresis before cDNA synthesis and sequencing (4, 5). However, viroids generally occur at low concentrations in the infected host, making viroid discovery a challenging task for many plant pathology laborato...