Single Nucleotide Level Research Articles

Genome-wide profiling of BK polyomavirus integration in bladder cancer of kidney transplant recipients reveals mechanisms of the integration at the nucleotide level.

Chronic BK polyomavirus (BKPyV) infection is recognized as a potential oncogenic factor of urothelial carcinoma (UC) in renal transplant recipients. Recent studies have reported a positive correlation among BKPyV integration, persistent overexpression of viral large T antigen (TAg), and malignancy, yet little is known about the specific integration mechanisms and the impacts of viral integration. Here, we performed whole-genome sequencing (WGS) and viral capture-based sequencing on high-grade immunohistochemically TAg-positive UCs in two renal transplant recipients. A total of 181 integration sites, including the three found by WGS, were identified by viral capture-based sequencing, indicating its enhanced sensitivity and ability in identifying low-read integration sites in subpopulations of the tumor cells. The microhomologies between human and BKPyV genomes were significantly enriched in the flanking regions of 84.5% the integration sites, with a median length of 7 bp. Notably, 75 human genes formed fusion sequences due to viral insertional integration. Among them, the expression of 15 genes were statistically associated with UC based on GEO2R expression analysis. Our results indicated a multisite and multifragment linear integration pattern and a potential microhomology or nonhomologous end joining integration mechanism at the single-nucleotide level. We put forward a potential selection mechanism driven by immunity and centered on viral integration in the carcinogenesis of BKPyV.

Detect differentially methylated regions using non-homogeneous hidden Markov model for bisulfite sequencing data.

DNA methylation plays an important role in many biological processes and diseases. With the rise of the whole genome bisulfite sequencing technique, aberrant methylation patterns can now be detected by comparing paired normal and disease samples at the single nucleotide level. We develop a novel Bayesian method for detecting differentially methylated regions from paired bisulfite sequencing data, and implement it as a R package called BSDMR. Based on a non-homogeneous hidden Markov model, BSDMR provides a better modeling strategy for the spatial correlation between CpG sites and takes into consideration the relationship between methylation signals from normal and disease samples. Simulations show that BSDMR performs well even under low read depth and has a smaller false discovery rates than existing methods. We also apply BSDMR to the colon cancer data from Gene Expression Omnibus. The detected DMRs are well supported by existing biomedical literatures.

Ori-Finder 3: a web server for genome-wide prediction of replication origins in Saccharomyces cerevisiae.

DNA replication is a fundamental process in all organisms; this event initiates at sites termed origins of replication. The characteristics of eukaryotic replication origins are best understood in Saccharomyces cerevisiae. For this species, origin prediction algorithms or web servers have been developed based on the sequence features of autonomously replicating sequences (ARSs). However, their performances are far from satisfactory. By utilizing the Z-curve methodology, we present a novel pipeline, Ori-Finder 3, for the computational prediction of replication origins in S. cerevisiae at the genome-wide level based solely on DNA sequences. The ARS exhibiting both an AT-rich stretch and ARS consensus sequence element can be predicted at the single-nucleotide level. For the identified ARSs in the S. cerevisiae reference genome, 83 and 60% of the top 100 and top 300 predictions matched the known ARS records, respectively. Based on Ori-Finder 3, we subsequently built a database of the predicted ARSs identified in more than a hundred S. cerevisiae genomes. Consequently, we developed a user-friendly web server including the ARS prediction pipeline and the predicted ARSs database, which can be freely accessed at http://tubic.tju.edu.cn/Ori-Finder3.

Recognition of Single‐Point Mutation Using a Biological Nanopore

AbstractHere the recognition of a single‐point mutation in oligonucleotides is described by using nanopore measurements. The translocation behavior of a series of mutated DNA strands, hybridized with a complementary DNA probe, is analyzed via blocking current and unzipping time. Discernment of the mutation position at the single nucleotide level is achieved by analysis of a 2D graph of the bootstrapped translocation data. The proposed approach provides a useful tool for the mutation detection of oligonucleotides secreted from tumor cells and is applicable in simple and label‐free diagnoses as a nanopore liquid biopsy.

A comprehensive evaluation of computational tools to identify differential methylation regions using RRBS data

DNA methylation plays a vital role in transcription regulation. Reduced representation bisulfite sequencing (RRBS) is becoming common for analyzing genome-wide methylation profiles at the single nucleotide level. A major goal of RRBS studies is to detect differentially methylated regions (DMRs) between different biological conditions. The previous tools to predict DMRs lack consistency. Here, we simulated RRBS datasets with significant attributes of real sequencing data under a wide range of scenarios, and systematically evaluated seven DMR detection tools in terms of type I error rate, precision/recall (PR), and area under ROC curve (AUC) using different methylation levels, sequencing coverage depth, length of DMRs, read length, and sample sizes. DMRfinder, methylSig, and methylKit were our preferred tools for RRBS data analysis, in terms of their AUC and PR curves. Our comparison highlights the different applicability of DMR detection tools and provides information to guide researchers towards the advancement of sequence-based DMR analysis.

Stress, Adaptation, and the Deep Genome: Why Transposons Matter.

Stress is a common, if often unpredictable life event. It can be defined from an evolutionary perspective as a force an organism perceives it must adapt to. Thus stress is a useful tool to study adaptation and the adaptive capacity of organisms. The deep genome, long neglected as a pile of "junk" has emerged as a source of regulatory DNA and RNA as well as a potential stockpile of adaptive capacity at the organismal and species levels. Recent work on the regulation of transposable elements (TEs), the principle constituents of the deep genome, by stress has shown that these elements are responsive to host stress and other environmental cues. Further, we have shown that some are likely directly regulated by the glucocorticoid receptor (GR), one of the two major vertebrate stress steroid receptors in a fashion that appears adaptive. On the basis of this and other emerging evidence I argue that the deep genome may represent an adaptive toolkit for organisms to respond to their environments at both individual and evolutionary scales. This argues that genomes may be adapted for what Waddington called "trait adaptability" rather than being purely passive objects of natural selection and single nucleotide level mutation.

Distinct Life Histories Impact Dikaryotic Genome Evolution in the Rust Fungus Puccinia striiformis Causing Stripe Rust in Wheat.

Stripe rust of wheat, caused by the obligate biotrophic fungus Puccinia striiformis f.sp. tritici, is a major threat to wheat production worldwide with an estimated yearly loss of US $1 billion. The recent advances in long-read sequencing technologies and tailored-assembly algorithms enabled us to disentangle the two haploid genomes of Pst. This provides us with haplotype-specific information at a whole-genome level. Exploiting this novel information, we perform whole-genome comparative genomics of two P. striiformis f.sp. tritici isolates with contrasting life histories. We compare one isolate of the old European lineage (PstS0), which has been asexual for over 50 years, and a Warrior isolate (PstS7 lineage) from a novel incursion into Europe in 2011 from a sexual population in the Himalayan region. This comparison provides evidence that long-term asexual evolution leads to genome expansion, accumulation of transposable elements, and increased heterozygosity at the single nucleotide, structural, and allele levels. At the whole-genome level, candidate effectors are not compartmentalized and do not exhibit reduced levels of synteny. Yet we were able to identify two subsets of candidate effector populations. About 70% of candidate effectors are invariant between the two isolates, whereas 30% are hypervariable. The latter might be involved in host adaptation on wheat and explain the different phenotypes of the two isolates. Overall, this detailed comparative analysis of two haplotype-aware assemblies of P. striiformis f.sp. tritici is the first step in understanding the evolution of dikaryotic rust fungi at a whole-genome level.

Evidence for re-infection and persistent carriage of Shigella species in adult males reporting domestically acquired infection in England

ObjectivesWe analysed national surveillance typing data of Shigella isolated from adult males with domestically acquired infection (a cohort largely consisting of men who have sex with men (MSM)) to establish whether multiple isolates from the same individual over time represented persistent carriage or re-infection. MethodsWe carried out a retrospective cohort study of adult males diagnosed with Shigella from 2004 to 2018. Median time intervals between multiple isolations of Shigella flexneri and S. sonnei were compared. Analysis of whole genome sequencing data provided strain discrimination at the single nucleotide level and was used to quantify the genetic distance among isolates. Maximum likelihood phylogenies were constructed to determine whether persistent carriage (characterized by multiple isolations of the same strain) or re-infection (characterized by multiple isolations of different strains) was best supported by the phylogenetic analysis. A comparison analysis was carried out using data linked to adult females with domestically acquired shigellosis. ResultsThe number of men reporting multiple isolations of Shigella species was 165/4733 (3.5%) compared with 31/2423 (1.3%) females (p < 0.001). For isolate pairs from men associated with persistent carriage, the isolation time interval range was 6–176 days (median 23.5; IQR 8–70) and single nucleotide polymorphism (SNP) distance range was 0–7 SNPs (median 0.5; IQR 0–2). For those associated with re-infection, the isolation time interval was 34–2636 days (median 732; IQR 191–1258) and the SNP distance was 10–1462 SNPs (median 120; IQR 29–377). DiscussionMultiple Shigella isolations in individuals with domestically acquired infections was more frequently observed in adult males than in adult females. Following the acute phase of infection, carriage can persist for months, and infection can recur within months, even with strains belonging to the same species and the same serotype. A combination of multiple sexual partners, persistent carriage following the acute phase of infection and evidence of recurrent re-infection is likely to contribute to sustained transmission in this population.

EpiMOLAS: an intuitive web-based framework for genome-wide DNA methylation analysis

BackgroundDNA methylation is a crucial epigenomic mechanism in various biological processes. Using whole-genome bisulfite sequencing (WGBS) technology, methylated cytosine sites can be revealed at the single nucleotide level. However, the WGBS data analysis process is usually complicated and challenging.ResultsTo alleviate the associated difficulties, we integrated the WGBS data processing steps and downstream analysis into a two-phase approach. First, we set up the required tools in Galaxy and developed workflows to calculate the methylation level from raw WGBS data and generate a methylation status summary, the mtable. This computation environment is wrapped into the Docker container image DocMethyl, which allows users to rapidly deploy an executable environment without tedious software installation and library dependency problems. Next, the mtable files were uploaded to the web server EpiMOLAS_web to link with the gene annotation databases that enable rapid data retrieval and analyses.ConclusionTo our knowledge, the EpiMOLAS framework, consisting of DocMethyl and EpiMOLAS_web, is the first approach to include containerization technology and a web-based system for WGBS data analysis from raw data processing to downstream analysis. EpiMOLAS will help users cope with their WGBS data and also conduct reproducible analyses of publicly available data, thereby gaining insights into the mechanisms underlying complex biological phenomenon. The Galaxy Docker image DocMethyl is available at https://hub.docker.com/r/lsbnb/docmethyl/.EpiMOLAS_web is publicly accessible at http://symbiosis.iis.sinica.edu.tw/epimolas/.

MASQC: Next Generation Sequencing Assists Third Generation Sequencing for Quality Control in N6-Methyladenine DNA Identification.

DNA N6-methyladenine (6mA) modification has been discovered as the most prevalent DNA modification in prokaryotes and eukaryotes, involving gene expression, DNA replication and repair, and host-pathogen interactions. Single-molecule real-time sequencing (SMRT-seq) can detect 6mA events in prokaryotic and eukaryotic genomes at the single-nucleotide level. However, there are no strict and economical quality control methods for high false-positive 6mA events in eukaryotic genomes. Therefore, by analyzing the distribution of 6mA in eukaryotic and prokaryotes, we proposed a method named MASQC (MeDIP-seq assists SMRT-seq for quality control in 6mA identification), which can identify 6mA events without doing the whole genome amplification (WGA) sequencing. The proposed MASQC method was assessed on two eukaryotic genomes and six bacterial genomes, our results demonstrate that MASQC performs well in quality control of false positive 6mA identification for both eukaryotic and prokaryotic genomes.

Genomes from around the globe

Human Genetics Genomic sequencing of diverse human populations to understand overall genetic diversity has lagged behind in-depth examination of specific populations. To add to our understanding of human genetic diversity, Bergstrom et al. generated whole-genome sequences surveying individuals in the Human Genome Diversity Project, which is a panel of global populations that has been instrumental in understanding the history of human populations. The authors' study adds data about African, Oceanian, and Amerindian populations and indicates that diversity tends to result from differences at the single-nucleotide level rather than copy number variation. An analysis of archaic sequences in modern populations identifies ancestral genetic variation in African populations that likely predates modern humans and has been lost in most non-African populations. Science , this issue p. [eaay5012][1] [1]: /lookup/doi/10.1126/science.aay5012

Depression phenotype identified by using single nucleotide exact amplicon sequence variants of the human gut microbiome.

Single nucleotide exact amplicon sequence variants (ASV) of the human gut microbiome were used to evaluate if individuals with a depression phenotype (DEPR) could be identified from healthy reference subjects (NODEP). Microbial DNA in stool samples obtained from 40 subjects were characterized using high throughput microbiome sequence data processed via DADA2 error correction combined with PIME machine-learning de-noising and taxa binning/parsing of prevalent ASVs at the single nucleotide level of resolution. Application of ALDEx2 differential abundance analysis with assessed effect sizes and stringent PICRUSt2 predicted metabolic pathways. This multivariate machine-learning approach significantly differentiated DEPR (n = 20) vs. NODEP (n = 20) (PERMANOVA P < 0.001) based on microbiome taxa clustering and neurocircuit-relevant metabolic pathway network analysis for GABA, butyrate, glutamate, monoamines, monosaturated fatty acids, and inflammasome components. Gut microbiome dysbiosis using ASV prevalence data may offer the diagnostic potential of using human metaorganism biomarkers to identify individuals with a depression phenotype.

Genome-based breeding approaches in major vegetable crops.

Vegetable crops are major nutrient sources for humanity and have been well-cultivated since thousands of years of domestication. With the rapid development of next-generation sequencing and high-throughput genotyping technologies, the reference genome of more than 20 vegetables have been well-assembled and published. Resequencing approaches on large-scale germplasm resources have clarified the domestication and improvement of vegetable crops by human selection; its application on genetic mapping and quantitative trait locus analysis has led to the discovery of key genes and molecular markers linked to important traits in vegetables. Moreover, genome-based breeding has been utilized in many vegetable crops, including Solanaceae, Cucurbitaceae, Cruciferae, and other families, thereby promoting molecular breeding at a single-nucleotide level. Thus, genome-wide SNP markers have been widely used, and high-throughput genotyping techniques have become one of the most essential methods in vegetable breeding. With the popularization of gene editing technology research on vegetable crops, breeding efficiency can be rapidly increased, especially by combining the genomic and variomic information of vegetable crops. This review outlines the present genome-based breeding approaches used for major vegetable crops to provide insights into next-generation molecular breeding for the increasing global population.

Evolution of Streptococcus pneumoniae Serotype 3 in England and Wales: A Major Vaccine Evader.

Despite its inclusion in pneumococcal conjugate vaccine 13 (PCV13), Streptococcus pneumoniae serotype 3 remains a major cause of invasive pneumococcal disease in England and Wales. Previous studies have indicated that there are distinct lineages within serotype 3 clonal complex 180 and the clade distributions have shifted in recent years with the emergence of clade II. We undertook whole genome sequencing and genomic analysis of 616 serotype 3 isolates from England and Wales between 2003 and 2018, including invasive and carriage isolates. Our investigations showed that clade II has expanded since 2014 and now represents 50% of serotype 3 invasive pneumococcal disease (IPD) isolates in England and Wales. Genomic analysis of antibiotic resistance and protein antigen genes showed that distinct profiles are present within the clades which could account for the recent emergence of this clade. This investigation highlights the importance and utility of routine whole genome sequencing and its ability to identify new and emerging variation at the single nucleotide level which informs surveillance and will impact future vaccine development.

1438MicroRNA editing is integral for interleukin-6 trans-signalling and leukocyte trafficking to ischemic tissues

Abstract Background/Aim Adenosine to inosine RNA editing is an essential post-transcriptional RNA modification catalysed by adenosine deaminase acting on RNA-1 and -2 (ADAR1; ADAR2). Endothelial cells (ECs) attract and guide leukocytes to sites of ischemic tissue injury. Here we studied the role of RNA editing in ischemic disease. Methods Primary human and murine vascular endothelial cell cultures were used to assess the EC responses to interleukin-6 (IL-6) or ischemia. For the animal studies, the effect of ADAR2 in acute and chronic ischemic disease was evaluated in cremaster muscle microcirculation by intravital microscopy, in peritoneal cavity after sterile peritonitis and in gastrocnemius muscle after hind-limb ischemia by 8-colour flow cytometry and immunohistochemistry (IHC) studies of Adar2−/−/tg as well as of i(nducible)EC-ADAR2 knockout (KO) mice. For the mechanistic studies, deep RNA sequencing, qRT-PCR, western blot, confocal microscopy, target-specific microRNA (miRNA) editing studies, RNA-immunoprecipitation, miRNA/plasmid silencing/overexpression and luciferase reporter assays were used among others. For human studies, ischemic tissues derived from patients with acute or chronic ischemic heart disease were processed. Results ADAR2, but not ADAR1, expression is induced by &gt;2-fold in hypoxic ECs and in ischemic vascular ECs in mice and humans. Unbiased gene ontology analysis of the EC transcriptome indicated that ADAR2 controls inflammatory responses and predominantly the expression of interleukin-6-signal transducer (IL6ST), the co-receptor of IL-6. Subsequently, ADAR2 controls IL-6 trans-signalling in ECs as documented by the STAT3 phosphorylation and expression of the downstream leukocyte adhesion molecules, E-selectin and VCAM-1. IL-6-inflamed cremaster muscles showed that rolling and adhesion of leukocyte subsets to vascular wall were severely impaired in Adar2−/−/tg mice. Leukocyte transmigration was also diminished by &gt;2-fold in Adar2−/−/tg and in iEC-ADAR2 KO mice in response to IL-6 or ischemia. Similar results were obtained for leukocyte rolling, adhesion and infiltration after acute (4h) and chronic (3d; 21d) ischemia from iEC-ADAR2 KO mice and human ischemic muscle tissues. Next we studied how ADAR2 controls IL6ST expression. ADAR2-deficient vascular EC miRNAome revealed the upregulation of a conserved group of miRNAs targeting the IL6ST mRNA including miR-199a-5p and miR-335-3p. At a single-nucleotide level, ADAR2-induced RNA editing of the stem loops of the primary miR-199a1/2 and miR-335 directly disrupted Drosha recruitment to both and thus inhibited their maturation process. Accordingly, rescue experiments using miRNA-inhibitors restored IL6ST levels after ADAR2 deficiency. Conclusion Taking together, inhibition of the microRNA maturation process by ADAR2-mediated RNA editing is integral for IL-6 trans-signalling in vascular endothelium and subsequent leukocyte trafficking to ischemic tissues in mice and humans.

Elimination of Reference Mapping Bias Reveals Robust Immune Related Allele-Specific Expression in Crossbred Sheep.

Pervasive allelic variation at both gene and single nucleotide level (SNV) between individuals is commonly associated with complex traits in humans and animals. Allele-specific expression (ASE) analysis, using RNA-Seq, can provide a detailed annotation of allelic imbalance and infer the existence of cis-acting transcriptional regulation. However, variant detection in RNA-Seq data is compromised by biased mapping of reads to the reference DNA sequence. In this manuscript, we describe an unbiased standardized computational pipeline for allele-specific expression analysis using RNA-Seq data, which we have adapted and developed using tools available under open license. The analysis pipeline we present is designed to minimize reference bias while providing accurate profiling of allele-specific expression across tissues and cell types. Using this methodology, we were able to profile pervasive allelic imbalance across tissues and cell types, at both the gene and SNV level, in Texel×Scottish Blackface sheep, using the sheep gene expression atlas data set. ASE profiles were pervasive in each sheep and across all tissue types investigated. However, ASE profiles shared across tissues were limited, and instead, they tended to be highly tissue-specific. These tissue-specific ASE profiles may underlie the expression of economically important traits and could be utilized as weighted SNVs, for example, to improve the accuracy of genomic selection in breeding programs for sheep. An additional benefit of the pipeline is that it does not require parental genotypes and can therefore be applied to other RNA-Seq data sets for livestock, including those available on the Functional Annotation of Animal Genomes (FAANG) data portal. This study is the first global characterization of moderate to extreme ASE in tissues and cell types from sheep. We have applied a robust methodology for ASE profiling to provide both a novel analysis of the multi-dimensional sheep gene expression atlas data set and a foundation for identifying the regulatory and expressed elements of the genome that are driving complex traits in livestock.

Multi-region exome sequencing reveals genomic evolution from preneoplasia to lung adenocarcinoma

There has been a dramatic increase in the detection of lung nodules, many of which are preneoplasia atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) or invasive adenocarcinoma (ADC). The molecular landscape and the evolutionary trajectory of lung preneoplasia have not been well defined. Here, we perform multi-region exome sequencing of 116 resected lung nodules including AAH (n = 22), AIS (n = 27), MIA (n = 54) and synchronous ADC (n = 13). Comparing AAH to AIS, MIA and ADC, we observe progressive genomic evolution at the single nucleotide level and demarcated evolution at the chromosomal level supporting the early lung carcinogenesis model from AAH to AIS, MIA and ADC. Subclonal analyses reveal a higher proportion of clonal mutations in AIS/MIA/ADC than AAH suggesting neoplastic transformation of lung preneoplasia is predominantly associated with a selective sweep of unfit subclones. Analysis of multifocal pulmonary nodules from the same patients reveal evidence of convergent evolution.

Impact of metal oxide nanoparticles on in vitro DNA amplification.

Polymerase chain reaction (PCR) is used as an in vitro model system of DNA replication to assess the genotoxicity of nanoparticles (NPs). Prior results showed that several types of NPs inhibited PCR efficiency and increased amplicon error frequency. In this study, we examined the effects of various metal oxide NPs on inhibiting PCR, using high- vs. low-fidelity DNA polymerases; we also examined NP-induced DNA mutation bias at the single nucleotide level. The effects of seven major types of metal oxide NPs (Fe2O3, ZnO, CeO2, Fe3O4, Al2O3, CuO, and TiO2) on PCR replication via a low-fidelity DNA polymerase (Ex Taq) and a high-fidelity DNA polymerase (Phusion) were tested. The successfully amplified PCR products were subsequently sequenced using high-throughput amplicon sequencing. Using consistent proportions of NPs and DNA, we found that the effects of NPs on PCR yield differed depending on the DNA polymerase. Specifically, the efficiency of the high-fidelity DNA polymerase (Phusion) was significantly inhibited by NPs during PCR; such inhibition was not evident in reactions with Ex Taq. Amplicon sequencing showed that the overall error rate of NP-amended PCR was not significantly different from that of PCR without NPs (p > 0.05), and NPs did not introduce single nucleotide polymorphisms during PCR. Thus, overall, NPs inhibited PCR amplification in a DNA polymerase-specific manner, but mutations were not introduced in the process.

SpliceV: analysis and publication quality printing of linear and circular RNA splicing, expression and regulation

BackgroundIn eukaryotes, most genes code for multiple transcript isoforms that are generated through the complex and tightly regulated process of RNA splicing. Despite arising from identical precursor transcripts, alternatively spliced RNAs can have dramatically different functions. Transcriptome complexity is elevated further by the production of circular RNAs (circRNAs), another class of mature RNA that results from the splicing of a downstream splice donor to an upstream splice acceptor. While there has been a rapid expansion of circRNA catalogs in the last few years through the utilization of next generation sequencing approaches, our understanding of the mechanisms and regulation of circular RNA biogenesis, the impact that circRNA generation has on parental transcript processing, and the functions carried out by circular RNAs remains limited.ResultsHere, we present a visualization and analysis tool, SpliceV, that rapidly plots all relevant forward- and back-splice data, with exon and single nucleotide level coverage information from RNA-seq experiments in a publication quality format. SpliceV also integrates analysis features that assist investigations into splicing regulation and transcript functions through the display of predicted RNA binding protein sites and the configuration of repetitive elements along the primary transcript.ConclusionsSpliceV is an easy-to-use splicing visualization tool, compatible with both Python 2.7 and 3+, and distributed under the GNU Public License. The source code is freely available for download at https://github.com/flemingtonlab/SpliceV and can be installed from PyPI using pip.

Reassessment of Viroid RNA Cytosine Methylation Status at the Single Nucleotide Level.

Composed of a few hundreds of nucleotides, viroids are infectious, circular, non-protein coding RNAs able to usurp plant cellular enzymes and molecular machineries to replicate and move in their hosts. Several secondary and tertiary RNA structural motifs have been implicated in the viroid infectious cycle, but whether modified nucleotides, such as 5C-methylcytosine (m5C), also play a role has not been deeply investigated so far. Here, the possible existence of m5C in both RNA polarity strands of potato spindle tuber viroid and avocado sunblotch viroid -which are representative members of the nucleus- and chloroplast-replicating viroids, respectively- has been assessed at single nucleotide level. We show that a standard bisulfite protocol efficiently used for identifying m5C in cellular RNAs may generate false positive results in the case of the highly structured viroid RNAs. Applying a bisulfite conversion protocol specifically adapted to RNAs with high secondary structure, no m5C was identified in both polarity strands of both viroids, indicating that this specific nucleotide modification does not likely play a role in viroid biology.