Early detection and improved genomic surveillance of SARS-CoV-2 variants from deep sequencing data

In the definition of fruitful strategies to contrast the worldwide diffusion of SARS-CoV-2, maximum efforts must be devoted to the early detection of dangerous variants. An effective help to this end is granted by the analysis of deep sequencing data of viral samples, which are typically discarded after the creation of consensus sequences. Indeed, only with deep sequencing data it is possible to identify intra-host low-frequency mutations, which are a direct footprint of mutational processes that may eventually lead to the origination of functionally advantageous variants. Accordingly, a timely and statistically robust identification of such mutations might inform political decision-making with significant anticipation with respect to standard analyses based on consensus sequences. To support our claim, we here present the largest study to date of SARS-CoV-2 deep sequencing data, which involves 220,788 high quality samples, collected over 20 months from 137 distinct studies. Importantly, we show that a relevant number of spike and nucleocapsid mutations of interest associated to the most circulating variants, including Beta, Delta and Omicron, might have been intercepted several months in advance, possibly leading to different public-health decisions. In addition, we show that a refined genomic surveillance system involving high- and low-frequency mutations might allow one to pinpoint possibly dangerous emerging mutation patterns, providing a data-driven automated support to epidemiologists and virologists.

RNA Editing and Its Roles in Plant Organelles

RNA editing, a vital supplement to the central dogma, yields genetic information on RNA products that are different from their DNA templates. The conversion of C-to-U in mitochondria and plastids is the main kind of RNA editing in plants. Various factors have been demonstrated to be involved in RNA editing. In this minireview, we summarized the factors and mechanisms involved in RNA editing in plant organelles. Recently, the rapid development of deep sequencing has revealed many RNA editing events in plant organelles, and we further reviewed these events identified through deep sequencing data. Numerous studies have shown that RNA editing plays essential roles in diverse processes, such as the biogenesis of chloroplasts and mitochondria, seed development, and stress and hormone responses. Finally, we discussed the functions of RNA editing in plant organelles.

Identification of Genetically Related HCV Infections Among Self-Described Injecting Partnerships

Background The current opioid epidemic across the United States has fueled a surge in the rate of new hepatitis C virus (HCV) infections among young persons who inject drugs (PWIDs). Paramount to interrupting transmission is targeting these high-risk populations and understanding the underlying network structures facilitating transmission within these communities. Methods Deep sequencing data were obtained for 52 participants from 32 injecting partnerships enrolled in the U-Find-Out (UFO) Partner Study, which is a prospective study of self-described injecting dyad partnerships from a large community-based study of HCV infection in young adult PWIDs from San Francisco. Phylogenetically linked transmission events were identified using traditional genetic-distance measures and viral deep sequence phylogenies reconstructed to determine the statistical support of inferences and the direction of transmission within partnerships. Results Using deep sequencing data, we found that 12 of 32 partnerships were genetically similar and clustered. Three additional phylogenetic clusters were found describing novel putative transmission links outside of the injecting relationship. Transmission direction was inferred correctly for 5 partnerships with the incorrect transmission direction inferred in more than 50% of cases. Notably, we observed that phylogenetic linkage was most often associated with a lower number of network partners and involvement in a sexual relationship. Conclusions Deep sequencing of HCV among self-described injecting partnerships demonstrates that the majority of transmission events originate from outside of the injecting partnership. Furthermore, these findings caution that phylogenetic methods may be unable to routinely infer the direction of transmission among PWIDs especially when transmission events occur in rapid succession within high-risk networks.

Generation of a Novel SARS-CoV-2 Sub-genomic RNA Due to the R203K/G204R Variant in Nucleocapsid: Homologous Recombination has Potential to Change SARS-CoV-2 at Both Protein and RNA Level

Background: Genetic variations across the SARS-CoV-2 genome may influence transmissibility of the virus and the host’s anti-viral immune response, in turn affecting the frequency of variants over time. In this study, we examined the adjacent amino acid polymorphisms in the nucleocapsid (R203K/G204R) of SARS-CoV-2 that arose on the background of the spike D614G change and describe how strains harboring these changes became dominant circulating strains globally. Methods: Deep-sequencing data of SARS-CoV-2 from public databases and from clinical samples were analyzed to identify and map genetic variants and sub-genomic RNA transcripts across the genome. Results: Sequence analysis suggests that the 3 adjacent nucleotide changes that result in the K203/R204 variant have arisen by homologous recombination from the core sequence of the leader transcription-regulating sequence (TRS) rather than by stepwise mutation. The resulting sequence changes generate a novel sub-genomic RNA transcript for the C-terminal dimerization domain of nucleocapsid. Deep-sequencing data from 981 clinical samples confirmed the presence of the novel TRS-CS-dimerization domain RNA in individuals with the K203/R204 variant. Quantification of sub-genomic RNA indicates that viruses with the K203/R204 variant may also have increased expression of sub-genomic RNA from other open reading frames. Conclusions: The finding that homologous recombination from the TRS may have occurred since the introduction of SARS-CoV-2 in humans, resulting in both coding changes and novel sub-genomic RNA transcripts, suggests this as a mechanism for diversification and adaptation within its new host.

Recovery of deleted deep sequencing data sheds more light on the early Wuhan SARS-CoV-2 epidemic

The origin and early spread of SARS-CoV-2 remains shrouded in mystery. Here I identify a data set containing SARS-CoV-2 sequences from early in the Wuhan epidemic that has been deleted from the NIH's Sequence Read Archive. I recover the deleted files from the Google Cloud, and reconstruct partial sequences of 13 early epidemic viruses. Phylogenetic analysis of these sequences in the context of carefully annotated existing data suggests that the Huanan Seafood Market sequences that are the focus of the joint WHO-China report are not fully representative of the viruses in Wuhan early in the epidemic. Instead, the progenitor of known SARS-CoV-2 sequences likely contained three mutations relative to the market viruses that made it more similar to SARS-CoV-2's bat coronavirus relatives.

IDENTIFICATION OF GENETICALLY RELATED HCV INFECTIONS AMONG SELF-DESCRIBED INJECTING PARTNERSHIPS

Background. The current opioid epidemic across the United States has fueled a surge in the rate of new HCV infections among young persons who inject drugs (PWIDs). Paramount to interrupting transmission is targeting these high-risk populations and understanding the underlying network structures facilitating transmission within these communities. Methods. Deep sequencing data were obtained for 52 participants from 32 injecting partnerships enrolled in the UFO Partner Study which is a prospective study of self-described injecting dyad partnerships from a large community-based study of HCV infection in young adult PWIDs from San Francisco. Phylogenetically linked transmission events were identified using traditional genetic-distance measures and viral deep sequence phylogenies reconstructed to determine the statistical support of inferences and the direction of transmission within partnerships. Results. Using deep sequencing data, we found that 12 of 32 partnerships were genetically similar and clustered. Three additional phylogenetic clusters were found describing novel putative transmission links outside of the injecting relationship. Transmission direction was inferred correctly for five partnerships with the incorrect transmission direction inferred in more than 50% of cases. Notably, we observed that phylogenetic linkage was most often associated with a lower number of network partners and involvement in a sexual relationship.

Reanalysis of deep-sequencing data from Austria points towards a small SARS-COV-2 transmission bottleneck on the order of one to three virions

An early analysis of SARS-CoV-2 deep-sequencing data that combined epidemiological and genetic data to characterize the transmission dynamics of the virus in and beyond Austria concluded that the size of the virus’s transmission bottleneck was large – on the order of 1000 virions. We performed new computational analyses using these deep-sequenced samples from Austria. Our analyses included characterization of transmission bottleneck sizes across a range of variant calling thresholds and examination of patterns of shared low-frequency variants between transmission pairs in cases where de novo genetic variation was present in the recipient. From these analyses, among others, we found that SARS-CoV-2 transmission bottlenecks are instead likely to be very tight, on the order of 1-3 virions. These findings have important consequences for understanding how SARS-CoV-2 evolves between hosts and the processes shaping genetic variation observed at the population level.

Molecular characterization of a soybean FT homologue, GmFT7

Soybean (Glycine max) is a vital oilseed legume crop that provides protein and oil for humans and feedstock for animals. Flowering is a prerequisite for seed production. Floral transition, from vegetative to reproductive stage, in a plant, is regulated by environmental (light, temperature) and endogenous factors. In Arabidopsis, Flowering Locus T (FT) protein is shown to be a mobile signal that moves from leaf to shoot apical meristem to induce flowering. However, FTs role in soybean is not fully resolved due to the presence of multiple (ten) homologs in the genome. Two of the ten FT homologs (GmFT2a and GmFT5a) have a role in the floral transition while GmFT1a and GmFT4 suppress soybean flowering. Recent deep sequencing data revealed that six FT homologs are expressed in shoot apical meristem and leaves during floral transition. One FT homolog, GmFT7 showed strong expression during soybean floral transition. Though bioinformatic analyses revealed that GmFT7 had high similarity with GmFT2a, ectopic GmFT7 expression in Arabidopsis could not promote flowering or rescue the late-flowering phenotype of Arabidopsis ft-10 mutant.

Case Report: Application of hepatitis B virus (HBV) deep sequencing to distinguish between acute and chronic infection

Deep sequencing of the full-length hepatitis B virus (HBV) genome provides the opportunity to determine the extent to which viral diversity, genotype, polymorphisms, insertions and deletions may influence presentation and outcomes of disease. Increasing experience with analysis of HBV genomic data opens up the potential for using these data to inform insights into pathophysiology of infection and to underpin decision making in clinical practice. We here set out to undertake whole genome HBV sequencing from an adult who presented acutely unwell with a new diagnosis of HBV infection, and tested positive for both HBV anti-core IgM and IgG, possibly representing either acute hepatitis B infection (AHB) or chronic hepatitis B with an acute reactivation (CHB-AR). The distinction between these two scenarios may be important in predicting prognosis and underpinning treatment decisions, but can be challenging based on routine laboratory tests. Through application of deep whole-genome sequencing we typed the isolate as genotype-D1, and identified several minority variants including G1764A and G1986A substitutions in the pre-core promoter and pre-core regions, which support CHB-AR rather than AHB. In the longer term, enhanced deep sequencing data for HBV may provide improved evidence to distinguish between acute and chronic infection, to predict outcomes and to stratify treatment.