Persons with disabilities (PwDs) face barriers in accessing sexual and reproductive health (SRH) services due to lack of knowledge and awareness, stigma and discrimination, and inadequate service provision. This study aimed to examine the determinants of SRH knowledge and awareness among PwDs in Ghana, and to explore their perceptions of the functionality of SRH services.A sequential explanatory mixed-methods study design was used to collect and analyse quantitative (n = 402) and qualitative (n = 37) data from PwDs in two districts in Ghana. Quantitative data were analysed using descriptive and inferential statistics, while qualitative data was analysed thematically.Most of the participants had high awareness of SRH (94.3%), sexually transmitted infections (STIs) (92.5%) and HIV/AIDS (97.0%). Employment status was positively associated with SRH awareness . Disability type was a significant predictor of STI  and HIV/AIDS  awareness, with the visually impaired having higher odds than the physically disabled. Age group was also a significant predictor of STI awareness, with older respondents having higher odds than younger ones . The qualitative findings revealed four main themes: conceptualisation of SRH, active engagement in SRH information seeking, tensions between knowledge and religious beliefs and perceived utility of SRH services.Despite the high levels of SRH knowledge and awareness among PwDs, there are significant gaps and challenges related to disability type, age group, misconceptions, beliefs, and service non-functionality that limit the utility of SRH services. The findings call for tailored education to reduce misconceptions and put in pragmatic steps to deliver quality SRH services and information to PwDs. Further research is needed to assess the sexual lives of PwDs and explore the perspectives of all relevant stakeholders, including service providers and policymakers on how to enhance SRH outcomes for PwDs in Ghana.The online version contains supplementary material available at 10.1186/s12978-023-01700-1. People with disability often encounter barriers in access sexual and reproductive health (SRH) services. These challenges arise from a lack of understanding and awareness about these services, stigma, and discrimination as well as unavailability of these services. This study sought to assess the extent of SRH knowledge and awareness among persons with disabilities (PwDs) in Ghana and to investigate their perceptions of the functionality of SRH services. To accomplish this, a study design that combined both quantitative and qualitative research methods was employed. The study collected and analysed data from 402 PwDs in two districts of Ghana. The quantitative data were subjected to descriptive and inferential statistical analyses, whereas the qualitative data, were thematically analysed. The findings indicated that a substantial number of participants demonstrated high awareness of SRH, including sexually transmitted infections (STIs) and HIV/AIDS. The participants' employment status was positively associated with their level of SRH awareness. Additionally, the type of disability played a significant role in predicting awareness of STIs and HIV/AIDS. Visually impaired individuals exhibited a greater likelihood of awareness compared to those with physical disabilities. Age also influenced STI awareness, with older respondents showing higher levels of awareness than their younger counterparts. Qualitative analyses unveiled four main themes: how SRH is conceptualised, the active seeking of SRH information, the tension between knowledge and religious beliefs, and the perceived usefulness of SRH services. The study concluded that PwDs have a high level of SRH knowledge and awareness. However, it identified gaps and challenges that stem from differences in disability type, age group, misconceptions, religious beliefs, and the effectiveness of the services provided. To address these issues, the study recommends tailored educational interventions to meet the unique needs and concerns of PwDs in relation to SRH. Furthermore, enhancing the quality and accessibility of SRH services for this population is suggested. The study underscores the need for further research on the sexual lives of PwDs and the views of stakeholders on how to enhance their SRH outcomes are needed.The online version contains supplementary material available at 10.1186/s12978-023-01700-1. According to the World Health Organization (WHO), 16% of the global population, or about one billion people, have some form of disability, and 80% of them live in low-and middle-income countries . Ghana hSRH is defined by the WHO as “a state of complete physical, emotional, mental, and social wellbeing in relation to all aspects of sexuality and reproduction, not merely the absence of disease, dysfunction, or infirmity” . SRH is STIs and HIV/AIDS are major global public health challenges that affect millions of people every year –14. TherSeveral policies and interventions have been introduced in Ghana to enhance the provision and accessibility of SRH services and information to the general population. These include the National Health Insurance Scheme , the Nat"Impact of health policies and interventions on the sexual and reproductive health outcomes among persons with disabilities in Ghana”. The framework describes how system characteristics, interventions, and client/individual characteristics all affect health outcomes or behaviours . ThFour experienced research assistants (RAs) from the Department of Population and Health, University of Cape Coast, Cape Coast and the Department of Health Promotion and Disability Studies, School of Public Health, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana were purposively recruited. They held bachelor and master degrees in the field of Population and Health, Disability and Rehabilitation as well as Geography. They had knowledge of disability issues and survey data collection. They underwent a five-day training using a designed training manual. The principal investigator (AS) trained them on the context, tool contents, and protocols for conducting successful interviews, including ethics and informed consent.The sample size for the quantitative phase was calculated using the formula by Lwanga, Lemeshow, and WHO , which yThe questionnaire , which were binary and functionality of SRH services. Functionality was how SRH services are meeting PwD’s needs. The respondents were asked to respond to a list of seven different statements on a 4-point Likert Scale The scale had a reliability score of 0.8049. The independent variables were the socio-demographic characteristics and disability-related factors . Braun and Clarke's  six stepThe socio-demographic characteristics of the 402 respondents are summarised in Table p < 0.001) (Table In Table 1) Table .Table 2PThe participants generally demonstrated high levels of awareness of SRH 94.3%), STIs (92.5%) and HIV/AIDS (97.0%) , whose ages spanned from 21 to 60 years. Predominantly sourced from the Kumasi Metropolis (n = 24), the participants were primarily affiliated with the Akan ethnic group (n = 31), adhered to the Christian faith (n = 34), and were married (n = 19). The rest of the participants were from the Offinso north district (n = 13) and various other ethnic backgrounds (n = 6).The thematic analysis of the data yielded four overarching themes comprising (a) conceptualisation of SRH, (b) engagement in SRH information seeking, (c) tensions between knowledge and religious beliefs (d) perceived utility of SRH services.. The issues discussed under this theme comprised PwDs description of what SRH entails and the various types of SRH commodities and services. From the data, both groups of PwDs, males and females from both districts demonstrated good understanding of SRH. They described SRH as involving measures to prevent unintended pregnancies, to prevent and treat STIs, to maintain menstrual hygiene, and to pursue a safe and healthy sexual life in general.“In my view, sexual and reproductive health is all about doing things that will protect you from sexually transmitted infections and unintended pregnancies. In terms of safe sex, you don’t have to have sex with every woman you meet. You have to make sure that you know the STI status of anyone you want to have sex with”. “I know that sexual and reproductive health is about family planning and contraceptive use so that you can easily space out your children and prevent sexually transmitted infections such as HIV/AIDS”. The first theme that emerged from the data was conceptualisation of SRH“When it comes to the type of family planning, I know about the implant that women insert in their arm. There is this girl here who did that, and I didn’t know. Whenever you hit her arm, you can feel like there is a metallic thing in there. So, she later confirmed that it was an implant. There are others who go for the oral contraceptive pills. I also know about the condom. If it comes to where people can get access to family planning commodities, they can go to the hospital or the pharmacy. For the condom and contraceptive pills, you can easily get it from the pharmacy”. “…Also, I am aware that SRH services include treatment and prevention of STIs such as Gonorrhoea. Previously, these diseases were only through sex but now, you can even contract some STIs such as candidiasis from the toilet facilities that we use. If you don’t want to get pregnant or get infected with any STI, then you can use a condom”. Participants also demonstrated knowledge of the various types of SRH commodities, including contraceptives and where to access these services. Furthermore, some participants exhibited awareness of some STIs such as Gonorrhea and Candidiasis.“I do research from Google and sometimes too I discuss it with my friends. Also, in school at the JHS and SHS level, they taught us this [SRH]. I also read books; I access Google to get more knowledge or information whenever I need to”. “I got this information [SRH] from the doctors who came to serve as facilitators for the health workshop. The union–XXX, invited the doctor from XXX to come and educate us on sexual and reproductive health. This happened in 2020, thus around the COVID-19 time. The time COVID-19 was really serious. Primarily, we get this information on SRH from the healthcare providers who are invited by the XXX. Apart from that, I have my radio where I receive a lot of information. Every Sunday, I tune my radio to listen to [XXX] who is at a government hospital at XXX at 5:30pm. He is a gynaecologist so all that he talks about are SRH and obstetric issues. So, he talks about family planning, birth control and so forth”. The second theme that emerged from the data was participants engagement in SRH information seeking. Participants reported various sources from which they obtained information about SRH. These sources included the media, disability organisations, HPs, extended family members, friends, and partners. The most commonly used source was mass media, which encompasses the internet, TV, and radio. For most participants, the media played a vital role in learning about SRH issues. The majority of the visually impaired were active information seekers from the radio.“It is something for those who are married. Sometimes, when you are married, you will want to space out your children. That is when the family planning comes through. You can go in for which ever will suit your system so that you can concentrate in life. We all know that sex is not for children, therefore, family planning is not for children”. “I think the family planning is good for those who have given birth before because I heard if you haven’t given birth and you do it consistently, it can cause infertility, or you might not be able to give birth”. Another major theme that emerged from the data was tensions between knowledge on SRH services and religious beliefs. Despite having knowledge and awareness of SRH services, some of the participants in both rural and urban areas held various misconceptions regarding the access and utilisation of certain SRH services and products. These misconceptions were exacerbated by their limited comprehensive knowledge of SRH and strong religious beliefs and doctrines opposing the use of contraceptives. Some participants expressed the view that family planning services should only be accessed by those who are married or already have children, and there was a belief that using family planning products could lead to side effects. Others also believed that the use of condoms reduces sexual pleasure.“…But in my view, it is sinful in the sight of God because using a condom is equivalent to aborting your pregnancy”. “…Also, I know about condoms. However, for that one, the Bible speaks against it. The Bible says that we should give birth and fill the earth. So, if you are using condoms, then how do you expect to give birth and fulfil this command by God. So, using condoms is very bad. I know that both family planning and condoms are the same because they both aim to prevent pregnancies. But in my view, if you abstain, that will be much effective at preventing STIs than using the condoms which is against the tenets of the Bible”. Even though most of the participants were aware of SRH services, religious beliefs also influenced their perceptions of some SRH products. Others equated the use of contraceptive services, such as condoms, to abortion. Some participants also discussed the tenets in the Bible regarding the need to give birth to more children to fulfill the responsibility God has imposed on humans to continue the existence of humanity on Earth.“…But I am not a fan of condoms because it is not pleasurable. It is just like eating a toffee with the wrapper on. So, I will never use a condom”. Other participants also indicated how the use of some SRH products diminishes sexual satisfaction.The fourth theme that emerged from the data was perspectives on utility of SRH services. The issues discussed under this theme were importance of SRH services and perception of functionality of SRH services.“These services help to prevent infections. For instance, using a condom can help prevent infections like syphilis and gonorrhoea. Then with the family planning, it can help us to effectively space out our pregnancies. Also, these sexual and reproductive health services encourage women to go for antennal care until they give birth”. “SRH services is good because it helps women to deliver safely. Doctors and health care providers have a lot of expertise and experience to handle pregnancy complications. And so, if you deliver at the hospital, they can easily detect when there is a problem with your pregnancy and provide you with the needed support. When I got pregnant, they [health care providers] assessed my condition and told me I needed to undergo a caesarean section. So, you don’t have to relent on antenatal care because it really helps to ensure safe delivery of your child”. Five main issues related to the importance of accessing SRH services were discussed. These included STIs prevention, prevention of unintended pregnancies, safe delivery, reduction in maternal mortality, and prevention of economic stress among couples. Most of the participants shared their knowledge on how access to SRH services, such as condoms, can prevent them from contracting STIs, including HIV/AIDS. Participants also demonstrated an understanding of the dual protection that condoms provide, providing protection against both unintended pregnancies and STIs. Some participants further discussed how access to SRH services can help reduce societal stigma.“I know that if I want to use family planning or buy any of the family planning commodities, I can go to the pharmacy shops. If you have an STI, you can go to the pharmacy, and they may have a small room where you can receive an injection to treat the disease. “If you only seek for SRH services, you will get it. It doesn’t matter your disability status. You will be provided family planning if you ask for it. No one can stop you from doing family planning”. “If I need information about condom or some contraceptives, I can easily move to the pharmacy shop and ask the pharmacist or the attendant there. So, in any drug store, you can get condom to buy. So, a disabled person can also easily go there to get some”. PwDs discussed the functionality of SRH as a continuum under availability, accessibility, and affordability. These issues also reflect the WHO  right to“I think that is easy to access SRH services as a PwD. However, for my colleagues who use a wheelchair they have to go to a health facility that is disability-friendly so that they can easily access the facility and services. I use the clutches and so that is not a challenge for me”. “I think to an extent the services are meeting the needs of PwDs. Although I said I got support from my partner, but anytime I attend hospital they take good care of me. And this made me to honour all my ANC appointments. So, it's functional”. The second issue that emerged from the participants' discussions on the functionality of SRH services concerned the accessibility of these services. While a few of the participants encompassing all demographic groups perceived that SRH services were accessible, the dominant view among the majority was that these services were inaccessible. The data also showed that, the type of disability and support from abled partners influence level of accessibility.“In some ways, it is okay. But it doesn’t really meet our needs because there are still challenges like how the healthcare providers talk to us. But because the services are available for us to access, we can manage that situation”. “I will say no they are not functional because people don’t regard PwDs. It is recently that the education has been intensified. When you go to the health facilities, it is not disability-friendly. If you don’t take care, you may not be able to enter the premises because of how the building is constructed. So, there is still more to be done to make them more functional”. “…But the truth of the matter is that PwDs always experience difficulties using some of these services. Talking about the transport system, I mean moving around when you are at the health facility and the nature of the beds. Sometimes you are struggling to climb but you need to force yourself and do it because you are not feeling well. So, I will say, they are not fully functional when you factor PwDs into the equation”. Conversely, PwDs from both rural and urban areas who also felt the SRH are inaccessible shared their views in the following quotes:“I remember that during my postnatal care visit to the hospital, we were given the option to choose any of the family planning methods and it will be provided to us for free”. “I was shy though, but the attendant was kind to me and did not discriminate against me. So, it was easy for me to get it. Moreover, the cost of the condom was not expensive for me to bear”. The third sub issue that emerged from the participants regarding the functionality of SRH was the affordability of the services. Although participants shared two contrasting views on the affordability of these SRH services, the prevalent view was that the high direct and indirect costs associated with accessing them were a barrier. The main challenge associated with this was the ineffectiveness of the NHIS, which makes it difficult for patients to obtain subsidised medications. The data also revealed that some SRH providers are friendly and supportive of the PwDs. Those who perceived the services to be affordable shared the following views:“The challenge is that some of the most major diseases are not covered by the national health insurance. Though it is there but sometimes when you are sick and needs certain medications that are not covered you must still use money to get those medicines. It just covers some minor medications. And this is a challenge because if they want it to really take care of the very poor then it should cover most of the diseases that are costly but when you go there, they will just give you some basic medications”. “Also, the distance to the hospital and the amount of money that you might end up paying at the hospital can be a disincentive for you to go to the health facility. When you are going to deliver at the hospital, you have to buy a lot of things. These things are very expensive. So, these things are major challenges especially when you don’t have money or people to support you financially”. However, the majority of PwDs irrespective of type of disability who expressed opposite views on the affordability of the SRH services stated the following opinions:Table This mixed-methods study aimed to assess the awareness and knowledge of PwDs regarding SRH and their perceptions of the functionality of SRH services in Ghana's Ashanti Region. The findings indicated that most participants were aware of SRH (94.3%), STIs (92.5%), and HIV/AIDS (97.0%). The findings also revealed that employment status, type of disability, and age group were associated with different levels of awareness of SRH, STIs and HIV/AIDS. Moreover, the study showed that PwDs generally perceived SRH services as dysfunctional in meeting their needs. The perception of the functionality of SRH services varied significantly by type of disability, level of education, and income level. The qualitative data complemented and enriched the quantitative data by providing insights into the meanings, misconceptions, and beliefs that PwDs had about SRH services.The qualitative data revealed that PwDs in both rural and urban areas understood SRH as encompassing various aspects, such as preventing unintended pregnancies, preventing, and managing STIs, and enjoying a safe and healthy sexual life. These meanings are in line with previous studies conducted in Ghana  and VietThe differences in the levels of awareness observed in this study compared to others could be attributed to several factors. First, access to SRH services may vary across different settings, as suggested by Mekonnen et al. . Second,The framework guiding this study also suggests that client characteristics influence various health outcomes –37. In lConsistent with previous studies in Ghana  and ChinAge was also significantly associated with knowledge and awareness of STIs in this study. PwDs aged 30–49 years were more likely to hear about STIs than their younger counterparts aged 18–29 years. This finding on the association between age and knowledge of STIs is in line with a previous study in Ghana . One reaAnother major theme that emerged from the data was tensions between knowledge and religious beliefs about SRH services among some participants across all demographics. Despite the knowledge and awareness on SRH services, some of the participants had various misconceptions about the access and use of some SRH services and products. These misconceptions were influenced by religious beliefs and doctrines that opposed the use of contraceptives. The influence of religion on SRH and health seeking behavior of PwDs has also been reported in previous studies in Ghana , 32. SomRegarding the functionality of SRH services, most PwDs who perceived SRH as dysfunctional interpreting it as not meeting their needs. In accordance with the health outcomes model –37, the The findings of this study have various policy and practice implications. First, SDGs 3.7 and 5.6 strive to achieve universal access to SRH information and services as well as the integration of SRH into national strategies and the realisation of universal access to SRH and rights, respectively . AlthougThe strength and limitations inherent in this study are worth discussing. The strength of this study lies in the use of a sequential explanatory mixed-methods design, which enabled the integration of both quantitative and qualitative research findings . This apIn conclusion, the study found relatively high awareness and knowledge about SRH services among PwDs. Type of disability was associated with awareness of STIs including HIV/AIDS. However, some misconceptions influenced by religious beliefs about SRH services persist. PwDs also perceived SRH service delivery as largely dysfunctional. The findings call for tailored education to reduce misconceptions and put in pragmatic steps to deliver quality SRH services and information to PwDs. Further studies could explore the sexual lives of PwDs and recommendations from stakeholders on the strategies to improve SRH outcomes among PwDs in Ghana.Additional file 1. Questionnaires for_PWDs.Additional file 2. STROBE_Checklist.Additional file 3. IDI_Guide_PWDs.Additional file 4. COREQ.


In this review, Putnam et al. tackle our current understanding of RNA granules, intracellular phase-separated sites of RNA metabolism that contain factors for RNA biogenesis and turnover and are often assumed to represent specialized compartments for RNA biochemistry. The authors discuss the possibility that some RNA granules are condensation by-products that form when subsoluble RNP complexes saturate the cytoplasm or nucleoplasm, and introduce the term “incidental condensates” to refer to condensates that are tolerated by cells but do not add functionality beyond that provided by the soluble pool of saturating RNP complexes. Finally, they consider best practices for distinguishing functional RNA granules from incidental condensates. RNA granules are mesoscale assemblies that form in the absence of limiting membranes. RNA granules contain factors for RNA biogenesis and turnover and are often assumed to represent specialized compartments for RNA biochemistry. Recent evidence suggests that RNA granules assemble by phase separation of subsoluble ribonucleoprotein (RNP) complexes that partially demix from the cytoplasm or nucleoplasm. We explore the possibility that some RNA granules are nonessential condensation by-products that arise when RNP complexes exceed their solubility limit as a consequence of cellular activity, stress, or aging. We describe the use of evolutionary and mutational analyses and single-molecule techniques to distinguish functional RNA granules from “incidental condensates.” RNA granules are intracellular RNA–protein assemblies not enclosed by membranes that range in size from ∼100 nm to several micrometers. RNA granules have been observed in animal, fungus, plant, and prokaryotic cells and fall into three general classes: ubiquitous, cell type-specific, and stress-induced. Over 20 types of RNA granules have been described so far, each with a unique composition, comprising in some cases dozens of proteins and thousands of RNAs .Proteins enriched in RNA granules function in many aspects of RNA metabolism, from transcription and processing in the nucleus to translation and RNA turnover in the cytoplasm. By extension, RNA granules are often assumed to represent functional compartments that house RNA-focused activities that require the granule environment . For exaRecent findings have linked RNA granule assembly to phase separation of RNA–protein (RNP) complexes . Phase scsat), the sum of favorable intermolecular interactions offsets the entropic cost of demixing, causing interacting molecules to redistribute (“phase separate”) into a dense phase (condensate) and a dilute phase  interactions and segregative (“repulsive”) interactions that cause molecules to sort into distinct phases above Box 1.Genetic studies have shown that sequence-specific protein-binding domains contribute to RNA granule assembly in cells. For example, oligomerization domains in Edc3, G3BP, and PGL-1 are required to assemble P-bodies, stress granules, and P granules, respectively, likely because these domains mediate the formation of subsoluble RNP complexes . In addicsat of RNA-binding proteins by functioning as a multivalent scaffold or raise csat by competing with protein–protein interactions . Consistent with these theoretical predictions, RNA granule assembly in cells has been correlated with changes in the concentration or valency of proteins and RNAs. For example, the polarized condensation and dissolution of P granules coincides temporarily with the formation of concentration gradients across the cytoplasm of the s zygote . The asss zygote . Some nus zygote . Condenss zygote . AlthougBox 2.Theoretical considerations indicate that multivalent polymers in initially liquid condensates will “harden” over time by maximizing interactions with their neighbors, leading to “kinetic trapping,” where molecules are unable to exit or enter the condensate . Experimcsat, condensates will dissolve (lose molecules) at a rate proportional to the rate at which molecules in the condensates liberate themselves from their neighbors to enter the dilute phase. Similarly, when placed in a concentrated environment above csat, condensates will grow on a time scale proportional to the rate at which molecules at the interface rearrange to accommodate new neighbors incoming from the dilute phase. These theoretical considerations generally mean that reducing binding interactions between condensate components will increase csat, increase the rate of disassembly in undersaturated conditions (c < csat), and increase the rate of growth in oversaturated conditions (c > csat).Condensate hardening is potentially problematic because slow dynamics extend the time scale at which condensates will respond to environmental perturbations. When placed in a dilute environment below Escherichia coli behaves like a glass-forming liquid that requires constant energetic input from metabolism to remain fluid, becoming “vitrified” under low energy conditions , redistribution of activity from the dilute phase to the condensates will be minimal.A common assumption is that molecules in RNA granules are highly concentrated in the granules and only active in the granule environment. This assumption has led to the widespread hypothesis that RNA granules represent functional compartments, akin to organelles, that house specialized functions not possible in the more dilute environment of the cytoplasm or nucleoplasm . Howevercsat by increasing valency and decreasing solubility as a function of size  and a fraction demixes into phase-separated condensates. If the condensates enrich active RNP complexes or change their activity, the condensates will represent functional compartments that house (or suppress) the activity associated with the RNPs localized therein. On the other hand, if the condensates do not create  new activity, the condensates will have no functional consequences. We refer to such nonfunctional condensates as “incidental” to denote the fact that their assembly is a secondary consequence of developmental, physiological, or stress-induced changes in the concentration, affinity, or valency of RNP complexes, leading to oversaturation of the cytoplasm or nucleoplasm . In the RNA granules typically have been assigned functions based on composition and/or biochemical experiments using condensates reconstituted in vitro . Six genDrosophila oocytes are translationally repressed prior to entering the granules. Third, mRNAs that enrich in P granules maintain low ribosome occupancy and translational repression even in embryos that lack P granules  drive highly efficient condensation of Pumilio, depleting the soluble pool by half, which in turn activates the translation of Pumilio mRNA targets  can be used to adjust conditions for reconstitutions in vitro to better match the in vivo setting. Parameters measured in vivo and in vitro in turn can inform theory to test and refine quantitative models e.g., . UltimatPhase separation of multivalent RNP complexes into condensates is an attractive model for the compartmentalization of RNA-focused activities in cells. For some native RNA granules, observations in cells are consistent with the physical properties of condensates, providing a sound theoretical framework for modeling RNA granule dynamics. Because the inside of phase-separated condensates is chemically and diffusionally distinct from the surrounding phase, condensates hold great promise as compartments with unique biochemistry. At the same time, the exquisite sensitivity of phase separation to small changes in solubility and concentration raises the possibility that some condensates are “incidental,” tolerated by cells as by-products of cellular activity but providing no new function.Although not functional, incidental condensates could still be useful to experimentalists as reporters of cellular activities involving subsoluble RNP complexes. For example, the sudden appearance of P-bodies signals the onset of maternal mRNA turnover in embryos . Analysi


To evaluate this claim, we compared the Agilent 4200 TapeStation’s RIN equivalent (RINe) and DV200 to the Agilent 2100 Bioanalyzer’s RIN for 183 parallel RNA samples. In our study, using RNA from a total of 183 human postmortem brain samples, we found that the RIN and RINe values only weakly correlate, with an r2 of 0.393 and an average difference of 3.2 RIN units. DV200 also only weakly correlated with RIN (r2 of 0.182) and RINe (r2 of 0.347). Finally, when applying a cut-off value of 6.5 for both metrics, we found that 95.6% of samples passed with RIN, while only 23.5% passed with RINe. Our results suggest that even though RIN  and RINe (TapeStation) use the same 1–10 value system, they should not be used interchangeably, and cut-off values should be calculated independently.The determination of RNA integrity is a critical quality assessment tool for gene expression studies where the experiment’s success is highly dependent on the sample quality. Since its introduction in 1999, the gold standard in the scientific community has been the Agilent 2100 Bioanalyzer’s RNA integrity number (RIN), which uses a 1–10 value system, from 1 being the most degraded, to 10 being the most intact. In 2015, Agilent launched 4200 TapeStation’s RIN equivalent, and reported a strong correlation of r The RIN (RNA integrity number) measured via the Agilent 2100 Bioanalyzer is widely accepted in the scientific community as the gold standard for objectively assessing a sample’s RNA quality for downstream applications. The Bioanalyzer is an automated microfluidics-based platform that separates RNA by molecular weight, and detects RNA using a fluorescent intercalated dye and fluorescent detecting laser. The results are displayed in a gel-like image for the visualization of the fragment size and distribution, accompanied by an electropherogram (peaks) for RIN visualization based on a RIN value from 1  to 10 (fully intact). A proprietary algorithm is assigned to the sample, reportedly making RIN easily reproducible between users [Previously, RNA quality was evaluated via performing gel electrophoresis followed by analysis of the 28S:18S ribosomal bands where RNA with a 28S:18S ratio of 2 or greater is considered to be of high quality. This method is still used, but is sample-demanding  and dependent on subjective human interpretation .2 = 0.936; median error < ±0.4 RIN units). In this study, we compared RINe and DV200 to the gold standard RIN, using the same set of human brain samples, to assess how well the different measurements aligned.In recent years, new instruments for measuring RNA integrity have been developed. Two of these measures, RINe  and DV200 (the percentage of RNA fragments larger than 200 nucleotides in size), both measured via the Agilent 4200 TapeStation, have become more widely used. The TapeStation offers increased throughput by analyzing 96 samples in a single run . Moreover, the manufacturer, Agilent, reports a high correlation between RINe and RIN (rt-test showed that the RIN and RINe were significantly different (p < 0.0001), with an average difference of 3.2 RIN units.For all the 183 samples, the RIN range was between 3 and 10, with an average of 8.8 ± 1.1, and a median of 9.1, whereas the RINe range was from 2.6 to 7.5, with an average of 5.6 ± 1.1 and a median of 5.8. DV200 ranged from 80.7–95.1%, with an average of 91.1% ± 2.6%, and a median of 91.8% . A two-tp < 0.0001), the correlation was weaker than previously reported by Agilent (r2 = 0.393 vs. 0.936) in their technical note titled “Comparison of RIN and RINe Algorithms for the Agilent 2100 Bioanalyzer and the Agilent 2200 TapeStation systems”. A linear regression comparing RIN to DV200 and RINe to DV200 were also statistically significant (p < 0.0001), but with weak correlations of r2 = 0.182 and r2 = 0.347, respectively . Applying a quality cut-off value of 70% DV200, established by Illumina for sequencing, 183/183 (100.0%) samples would meet this standard  samples would be considered fit for downstream applications, whereas only 43/183 (23.5%) would be fit using the RINe, and these proportions are statistically significantly different  and fluorometer (Qubit) analyses are common and relatively affordable options for quickly analyzing RNA quality.The Nanodrop measures the absorbances of the nucleic acids (260 nm) relative to contaminants, such as protein (280 nm), or common impurities from RNA purification, such as phenol (230 nm). It provides a good estimate of the RNA concentration and purity. However, the Nanodrop does not discriminate between RNA and genomic DNA (gDNA), which also absorbs at 260 nm.The Qubit fluorometer assesses RNA quality via the assay Qubit RNA IQ, which uses two dyes. One dye binds to large intact RNA, and the other binds to small, degraded RNA fragments. The Qubit provides a number between 1 and 10 as an assessment of quality. Similar to the Nanodrop, the fluorescent dye used by the Qubit is not specific to RNA, and can also bind with gDNA present in the sample, which may contribute to an overestimate of the RNA concentration. Therefore, while both the Nanodrop and Qubit are valuable tools for assessing RNA quality, it is important to be aware of their limitations, especially regarding the potential interference from gDNA when interpreting the results.Samples can be analyzed via gel electrophoresis, allowing the 28S:18S ribosomal RNA ratio to be determined, with the visualization of both gDNA contamination and degraded RNA. Typically, a 28S:18S ratio of 2:1 is considered indicative of good quality [The TapeStation and Bioanalyzer have the same sample requirements for their respective assays, the RNA Screen Tape and RNA 6000 Nano kit. Both instruments require a minimum sample volume of 1μL, and the quantitative range for each is 25–500 ng/μL. In our comparison, the same volume and concentration of RNA were used for each instrument, and were within the established quantitative ranges. We did not use any samples with concentrations below 25 ng/μL, from which the assays used are unable to calculate reliable RNA integrity values. Additionally, for samples that have abnormal electropherogram traces for the expected sample type, the software will not provide a measurement, and will instead display a non-applicable (NA) reading. All samples used in this study were assigned a RIN and RINe value.Although the instruments appear similar in their analysis, they are ultimately based on different algorithms. The TapeStation measures the relative ratio of the degraded products in the fast zone to the 18S peak signal, where the fast zone is defined as the region between small RNAs and the 18S. As the total RNA degrades, the 18S and 28S rRNA peaks slowly disappear, and degradation products emerge in the fast zone. On the other hand, the Bioanalyzer assesses the entire electrophoretic trace, including the 28S and 18S peak ratios, their separation, and the presence or absence of degraded products.Our results suggest that there may be larger discrepancies between RIN and RINe than previously reported, emphasizing that these measurements should not be used interchangeably. Instead, separate quality thresholds should be established for each metric. This is an important observation because, currently, the broader scientific community may be working under the assumption that these methods are virtually equivalent. A general premise in the field is that RIN values lower than 6.5 are not considered adequate for downstream applications. If the same standards were applied to RINe, many RINe samples would fail quality control (QC) that they would pass with RIN. This was seen in our study, where only 23.5% of samples would pass QC using RINe, but 95.6% of samples would pass QC using RIN.Several explanations may be put forward to explain the discrepancies between our study and previously published results. The manufacture’s comparison of RIN and RINe included commercially prepared cell lines, as well as RNA from humans, rats, and mice. Agilent degraded the total RNA, by heating for various durations (0 to 120 min), to compare samples with a lower RNA integrity. The RIN values ranged from 1.2 to 10. These methods are further described in Agilent’s Technical Overview “Comparisons of RIN and RINe Algorithms for the Agilent 2100 Bioanalyzer and the Agilent 2200 TapeStation System”. In our comparison of RIN and RINe, we only analyzed RNA from postmortem human brain samples, and did not deliberately degrade the RNA to expand the range of RNA integrity; therefore, we had few samples with poor RNA integrity to compare. In this study, the highest values obtained for RIN extended up to the maximum of 10, while the maximum RINe obtained was 7.5. We hypothesize that the type of degradation that occurs due to normal postmortem processing delays is different than the deliberate heat denaturation after RNA Isolation that Agilent performed, and this could explain some of the differences in results. It is also possible that the TapeStation’s algorithm is more sensitive to the type of degradation that occurs during normal postmortem processing, while their reported high values of RINe 9 and 10 were achieved using commercially prepared cell lines.Considering that RINe continues to gain popularity as a quality assessment, more research still needs to be conducted to better assess what constitutes an appropriate quality threshold for this measure. One recent study indicated that DV200 is a better predictor than RINe for next-generation sequencing (NGS) success when both measurements were correlated with NGS library yields . This wawww.brainandbodydonationprogram.org, accessed on 6 September 2023), a program dedicated to the longitudinal clinicopathological study of neurodegenerative diseases and normal aging [The frozen human tissue samples used in this study were collected as part of the Arizona Study of Aging and Neurodegenerative Disorders (AZSAND), and the Brain and Body Donation Program  were used to extract RNA from 25 mg of frozen cerebellum, following a previously published protocol ,13. The A volume of 1μL of undiluted RNA from each sample was run in parallel on the Agilent 4200 TapeStation and 2100 Bioanalyzer, using the RNA screen tape  and RNA 6000 Nano kit , respectively. The concentrations of the samples ranged from 37.3 ng/μL to 476.3 ng/μL, and were within the quantitative range for both assays (25–500 ng/μL). RIN was recorded from the Bioanalyzer while RINe and DV200 were recorded from the TapeStation 4200. RIN and RINe are assigned to the sample automatically. DV200 is the percent of sample that is above 200 nucleotides, and is determined post-assay via entering 200 as the lower limit in the regional settings of the TapeStation 4200 software. The details on calculating the DV200 can be found in Illumina’s technical note on Evaluating RNA Quality from Formalin-Fixed Paraffin-Embedded (FFPE) Samples. Both RIN and RINe are expressed as a number between 1 and 10, while DV200 is expressed as a percentage. A cut-off value of RIN 6.5 and DV200 of 70% were used to determine how many samples would be suitable for most downstream applications.t-test, were performed using GraphPad Prism v.5  to assess the relationships between RIN, RINe, and DV200 and to compare their values. The statistical significance level used for each was p < 0.05. The DV200 expressed out of 100% was divided by a factor of 10, to obtain the same maximum score as RIN and RINe, for better visual comparison.Statistical analyses, including a linear regression and paired


Emaravirus, family Fimoviridae. There are at least two other non-classified species that may be added. Some of these viruses are spreading rapidly and cause economically important diseases on several crops, raising a need for a sensitive diagnostic technique for taxonomic and quarantine purposes. High-resolution melting (HRM) has shown to be reliable for the detection, discrimination, and diagnosis of several diseases of plants, animals, and humans. This research aimed to explore the ability to predict HRM outputs coupled to reverse transcription-quantitative polymerase chain reaction (RT-qPCR). To approach this goal a pair of degenerate genus-specific primers were designed for endpoint RT-PCR and RT-qPCR-HRM and the species in the genus Emaravirus were selected to framework the development of the assays. Both nucleic acid amplification methods were able to detect in-vitro several members of seven Emaravirus species with sensitivity up to one fg of cDNA. Specific parameters for in-silico prediction of the melting temperatures of each expected emaravirus amplicon are compared to the data obtained in-vitro. A very distinct isolate of the High Plains wheat mosaic virus was also detected. The high-resolution DNA melting curves of the RT-PCR products predicted in-silico using uMeltSM allowed saving time while designing and developing the RT-qPCR-HRM assay since the approach avoided extensive searching for optimal HRM assay regions and rounds of HRM tests in-vitro for optimization. The resultant assay provides sensitive detection and reliable diagnosis for potentially any emaravirus, including new species or strains.Twenty-four species of RNA viruses contain members infecting economically important crops that are classified within the genus  Emaravirus  is a group of negative-sense ssRNA viruses created in 2011. E. sorbi, with European mountain ash ringspot-associated virus (EMARaV) as the member, was the first species characterized and classified in the genus ), 3.2 mM free [Mg 2+], and 0% DMSO. The thermodynamic library was Blake & Delcourt 1998 [in-silico and in-vitro. The σ factor defines the cooperativity of the system that enables the denaturation of DNA strands.°Tm profiles were predicted for the RT-qPCR product sequences of HPWMoV 07–961, RRV, RYRSaV, EMARaV, and FMV  using uMeltequences . The uMeurt 1998 . The denin-silico for 13 Emaravirus species after a Primer-BLAST search  within a range of six°Ta, from 45.2°C to 58.6°C , with anThe first multiple detection assay  was performed in our laboratory at Oklahoma State University, USA. HPWMoV, RRV, EMARaV, FMV dS, FMV F3, and FMV 1B were detected. PPSMV1 and PPSMV2 were not detected see . The secThe LoD of endpoint RT-PCR and RT-qPCR-HRM with primers EMARA F7/R8 was 1 fg using the positive controls HPWMoV, RRV, and FMV 1B, except for EMARaV . The LoDPrimers EMARA F7/R8 were found specific to the emaraviruses tested. Positive controls HPWMoV, RRV, and RYRSaV were exclusively detected . Few faiin-vitro was validated by sequencing their RT-PCR products. All the viruses tested had high identity at the nucleotide level (>90%) to homolog accessions deposited in the GenBank, except HPWMoV-07-961, which showed lower identity. EMARaV-EM1 (KX397601) was 96.8% identical to EMARaV (AY563040). The amplified RT-PCR products of FMV isolates 1B, dS, and F3  were 94.6%, 94.6%, and 94% identical respectively to FMV (AM941711), and showed 98% identity among each other. The RT-PCR product of HPWMoV-07-961 (KX397605) showed 74.4% identity compared to HPWMoV-W1 (KT970499). The HPWMoV reference positive control (KX397606) had 96.4% identity to the other three reference isolates of HPWMoV: GG1 (KT988869), KS7 (KT988860), and Nebraska (KJ939623). The alignment between the two isolates of HPWMoV (07–961 and reference positive control) showed 77.3% nucleotide identity between each other. RRV-Oklahoma 1 (KX397607) had 97.32% identity with two isolates of RRV: WA2017 (MH581220) and BA2018 (MH581213) from California, USA. RYRSaV-Oklahoma (KX397607) had a 93.3% identity to RYRSaV (JF795479). For the construction of the ML phylogenetic tree of the emaravirus RdRp gene, LG+G+I [The identity of the emaraviruses tested GG1 KT9889, KS7  selected to 0.184407. When the predicted HRM°Tm was calculated without adjusting σ, the in-silico°Tm differed 1.86–3.51°C from the actual in-vitro°Tm. However, when σ was adjusted, the difference between°Tm in-silico and in-vitro was substantially reduced. The higher the GC content, the higher the°Tm, although a few°Tm values were larger than expected (uMeltequences . Table 2in-silico (predicted) which were compared with in-vitro  obtained data using the conserved RdRp partial region of emaraviruses. For this purpose, a primer set compatible with two chemistries, endpoint RT-PCR and RT-qPCR-HRM, was designed and tested. The results of the melting temperatures obtained for emaraviruses are shown in SM, which facilitated the selection of the diagnostic region and primers in-silico. Testing expected amplicon sequences (including its primer sequences) in-silico by uMeltSM facilitated assembling RT-qPCR with HRM, allowed saving time during assay design, and avoided repeated experiments of HRM in-vitro searching for the optimal diagnostic region. Previously, [Emaravirus.This study explores and addresses software-assisted prediction of melting curves of RT-qPCR products viously, , 57 repoin-silico. The amplified RT-qPCR-HRM products were distinguishable from weak non-specific artifacts due to differences in°Tm: 72–77°C for artifacts, in contrast to°Tm over 78°C for emaraviruses  and three viruses frequently found co-infecting wheat and rose . Weak noaviruses .in-vitro in our lab (Oklahoma State University) by RT-qPCR-HRM. PPSMV 1 and 2 tested negative  the addition of a custom-designed 5`G/C flap to the EMARA R8 primer , 59, 60 Emaravirus rules a 25% difference in the amino acid sequences of RdRp, GP, or NC [Emaravirus species. HPWMoV 07–961 clustered with HPWMoV isolates, but branched out from the HPWMoV clade and formed a monotypic lineage  the normalized data derived from plots of raw data as shown in Figs 2. In our experiments, this inverse correlation was attenuated with 4 mM MgCl2 in the reaction  of the°Tm of the less concentrated cDNA (i.e. from 100 fg– 1 fg). These increments in melting temperature were detected from pg toward fg . This°Tmreaction  and up tin-silico and in-vitro°Tm values. This adjustment more accurately fits the Blake and Delcourt model [2, cDNA, and dye. However, since high mutation rates may be common during replication of some RNA virus species, the assumption of Tm as a single species descriptor during diagnosis is discouraged. Single or dual point mutations occurring in the target may affect the melting curve pattern of the amplicon in the RT-qPCR-HRM assay. Yet, discriminatory tendencies and differences among species will continue to be evident and can be corroborated using biological repeats of the RT-qPCR-HRM assays, a broad number of plant specimens, and viral reference isolates geographically characterized.The cooperativity parameter (σ) was adjusted to better concert rt model  and the in-silico. The use of uMeltSM for the prediction of Tm of diagnostic genomic sequences allowed detection, precise discrimination, and time gaining during assay design since avoids the need for rounds of HRM tests in-vitro or streaming routine electrophoresis. However, mutations occurring during virus replication, circumspect the assumption of°Tm as a single species descriptor during diagnosis. Diagnosticians may face difficult diagnoses if dealing with small HRM°Tm differences. Therefore, predicting PCR-product regions with larger Tm differences among amplicons allows to unmistakably discriminate species.In summary, we explored and discussed parameters and criteria to predict and design sensitive, accurate, and reliable diagnostics compatible with two chemistries RT-PCR and RT-qPCR-HRM applied for detection and discrimination of several emaraviruses. The described method allowed the detection of five emaravirus members and discrimination among isolates of the same virus species. Detection of PPSMV 1 and 2 by our proposed emaravirus assays may need further validation. The herein predicted and described RT-qPCR-HRM has the potential to detect candidate emaraviruses including new species or strains that were tested S1 FigThe alignment of EMARA F7 corresponds to the anti-sense strand of the emaraviruses genome, and EMARA R8 corresponds to the sense strand. The underlined sequence in EMARA R8 shows a customized 5’ non-complementary sequence. Stars above the alignment indicate conserved positions. EMARA F7 and EMARA R8 align to the nucleotide regions 4532–4563 and 4830–4803, respectively, in the anti-sense strand genome of the European mountain ash ringspot-associated virus (AY563040). Both primers are in the Bunya_RdRp conserved domain (PFAM: PF04196) in the emaravirus RdRp gene.(TIF)Click here for additional data file.S2 FigThe detection limit of both assays was 1 fg using cDNA from infected plant tissue.(TIF)Click here for additional data file.S3 FigSM.The plot was calculated using uMelt(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableThe outputs retrieved 13 Emaravirus species. The meaning of the virus acronyms is in the main text.(DOCX)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


As disease patterns shift with changing demographics and climate, developing comprehensive and scalable tools for tracking and identifying emerging pathogens is an increasingly critical component in mounting a robust public health response. Metagenomic next-generation sequencing (mNGS) provides unbiased detection of etiological agents from diverse types of clinical specimen and has great potential to aid in the prediction and prevention of future outbreaks. However, available protocols are unsuitable for widespread and routine use, due to high cost and extended hands-on time required for sample processing. Thus there is a need for faster, cheaper, and more automatable sequencing methods that retain the accuracy and breadth of current protocols.Here we develop Pooled RNA Metagenomics; a cost-effective and highly-multiplexed method that accelerates the processing of RNA samples by barcoding individual samples early in the pipeline allowing for parallel preparation. We benchmark this method by comparing the output from clinical samples to that from other established mNGS methods. Additionally, the protocol is designed to preserve the RNA sense information in the resulting sequencing data, and we incorporate unique molecular identifiers upstream of amplification; two features that enhance the utility and flexibility of the method for downstream applications.Pooled RNA Metagenomics OverviewSchematic overview of the pooled mNGS workflow: RNA samples are ligated to uniquely barcoded adapters before being pooled together for cDNA synthesis with UMI incorporation and subsequent library construction PCR.We found that early pooling of barcoded RNA samples can reduce reagent costs by up to 80% and processing time by up to 60%. We demonstrate that pooling up to 32 samples in a single well results in similar recovery of complete viral genomes to those produced by other mNGS methods where each RNA is processed separately.Pooled RNA Metagenomics provides an innovative, cost-effective, scalable, and pathogen-agnostic approach to outbreak monitoring and prevention. The high throughput enabled by this method can be augmented by incorporating automation, which in tandem increases speed and scale. The protocol is also amenable to miniaturization, which can further reduce costs and is of particular interest for low input clinical specimens. In summary, Pooled RNA Metagenomics dramatically lowers the barrier to high throughput mNGS, making it a viable option for enhanced pathogen surveillance.Pardis C. Sabeti, MD, PhD, Danaher Corp: Board Member|Danaher Corp: Stocks/Bonds|Delve Bio: Advisor/Consultant|Delve Bio: Stocks/Bonds|NextGen Jane: Advisor/Consultant|Sherlock Biosciences, Inc: Advisor/Consultant|Sherlock Biosciences, Inc: Stocks/Bonds


Many biologically important RNAs fold into specific 3D structures conserved through evolution. Knowing when an RNA sequence includes a conserved RNA structure that could lead to new biology is not trivial and depends on clues left behind by conservation in the form of covariation and variation. For that purpose, the R-scape statistical test was created to identify from alignments of RNA sequences, the base pairs that significantly covary above phylogenetic expectation. R-scape treats base pairs as independent units. However, RNA base pairs do not occur in isolation. The Watson-Crick (WC) base pairs stack together forming helices that constitute the scaffold that facilitates the formation of the non-WC base pairs, and ultimately the complete 3D structure. The helix-forming WC base pairs carry most of the covariation signal in an RNA structure. Here, I introduce a new measure of statistically significant covariation at helix-level by aggregation of the covariation significance and covariation power calculated at base-pair-level resolution. Performance benchmarks show that helix-level aggregated covariation increases sensitivity in the detection of evolutionarily conserved RNA structure without sacrificing specificity. This additional helix-level sensitivity reveals an artifact that results from using covariation to build an alignment for a hypothetical structure and then testing the alignment for whether its covariation significantly supports the structure. Helix-level reanalysis of the evolutionary evidence for a selection of long non-coding RNAs (lncRNAs) reinforces the evidence against these lncRNAs having a conserved secondary structure. In this manuscript, I introduce a new measure of significant structured RNA covariation at helix level. I show that helix-level covariation is more sensitive and robust than that obtained at base pair level in order to identify evolutionarily conserved RNA structure. Helix-level covariation exposes the circularity of using proposed RNA structures to build alignments in which to test for structural covariation. Helix-level covariation reinforces the evidence against some lncRNAs with proposed structures having a conserved secondary structure. The identification of conserved RNA structure is an important tool to discover RNA transcripts performing biologically relevant and novel functions. Conserved RNA structure results in covariation observed between base paired positions in alignments of homologous sequences. Because a conserved RNA structure is not the only source of covariation, a statistical test is required to distinguish covariation due to RNA structure from covariation due to other sources. Because the space of RNA transcripts without an assigned function is large, it is important to build robust methods that provide stringent control of false positive rates.To that effect, R-scape , 2 impleR-scape calculates covariation between all pairs of position in an alignment, and p-values are obtained by treating pairs as independent from each other. However, RNA base pairs, especially Watson-Crick base pairs  do not occur in isolation, but they stack on each other forming helices of variable length, typically ranging from 3 to 25 base pairs with a median of 12 in Rfam 14.9 consensus structures . As it has been observed before  follows the uniform distribution Un.The fact that p-values following the null hypothesis are uniformly distributed is part of a more general argument. For any hypothesis H, described by a continuous probability distribution FH(X) is given byFH(X) is Un, and pn = 1 − FH(pn) is also Un.I show that by observing that the CDF on I considered four statistics to combine base pair p-values. Those have been selected because of their simplicity and mathematical convenience as they are all based on statistics that have closed-form distributions from which I can easily calculate an aggregated p-value. Those four p-value aggregation methods are:Fisher method  distributed variable follows an exponential distribution, that after scaling by −2 follows a df = 2N degrees of freedom.Then, a Fisher combined p-value is calculated asLancaster method  A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Marc Robinson-RechaviAcademic EditorPLOS Computational BiologyArne ElofssonSection EditorPLOS Computational Biology***********************ploscompbiol@plos.org immediately:A link appears below if there are any accompanying review attachments. If you believe any reviews to be missing, please contact Reviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: In this article, the author introduces an update to the R-Scape software package. Previous versions of R-Scape utilized a statistical test to determine the covariance of base paired RNA structures in the presence of variation . This most recent update expands upon this idea, aggregating base pairs into helix-level structures and providing a measurement of their covariance. Overall, this was an excellent manuscript that presents a very useful new approach for evaluating the evolutionary impact of RNA structure on sequence and for aiding our understanding of RNA structure/function. The author performed extensive testing of the new version of R-scape including and in-depth analysis of lncRNA structure, which provides further evidence in support of her previous findings regarding the significance of proposed covariation in lncRNAs. I think this is important work as it pushes for more rigor and care in the use of evolutionary arguments for RNA structure/function and will be of wide interest to the readership of PLoS Computational Biology. I have only a few comments below.The author argues provides evidence that the new helix aggregated metrics are more robust than the base-pair metrics. It would be helpful if they could provide suggestions or guidance to readers on what the roles of base pair vs helix level metrics should be: i.e., should each metric be calculated and assessed or are helix level metrics to replace base pair level ones? Are there cases where base pair level metrics could be better?When the author writes, “I observe that all methods except for Šidák report higher sensitivity for the same number of false positives than taking as supported all helices with at least one covarying base pair.” do they mean that the number of false positives doesn't change with different values of k but sensitivity does, or that sensitivity divided by false positives grows?Minor:The manuscript is heavily written in first-person, maybe “depersonalize” it a little bit.Figure 2  has a space but  does notThe light blue font is a bit hard to read on Fig. 2.A few noted minor grammar issues: e.g.“The Lancaster weights allow (one) to incorporate additional”“four aggregation[s] methods”“Two structure[s] ha(ve) been proposed”"explore a[n] structural function"Reviewer #2: In Elena Rivas PhDs' research article, "RNA covariation at helix-level resolution for the identification of evolutionarily conserved RNA structure," she presents a novel methodology for recognizing RNA structures that have been evolutionarily conserved. The paper highlights a new technique for assessing statistically significant covariation at the RNA helix level, which is achieved by combining the significance and power of covariation calculated at the base pair level. Rivas begins by discussing R-scape, a tool developed to identify base pairs in RNA sequence alignments that covary beyond phylogenetic expectation. R-scape traditionally treats base pairs as independent entities, but Rivas highlights the fact that RNA base pairs do not exist in isolation—they stack together to form helices, which together create the complete structure of the RNA. It's these helix-forming Watson-Crick base pairs that are believed to carry most of the covariation signal in an RNA structure.Introducing a new covariation measure that aggregates these signals at the helix level, Rivas compares its performance to the conventional base-pair level approach. She demonstrates that this new technique improves sensitivity in detecting evolutionarily conserved RNA structure without compromising specificity. The paper then discusses the concept of p-value aggregation, where a group of base pairs with borderline covariation evidence may collectively yield a more robust helix signal. This is achieved by aggregating the p-values of individual marginally significant pairs. Rivas asserts that this aggregation process doesn't decrease sensitivity; even a single base pair with a strong covariation signal can make the entire helix significant, irrespective of the significance of other base pairs in the helix.Rivas also establishes a benchmark to evaluate the performance of the helix-level significant covariation test. This benchmark includes real structural RNAs, synthetic structural RNAs, and decoy RNA alignments that lack structure. The results of the R-scape covariation analysis are combined and ranked by helix E-value, and the ranked list of helices is used to plot the fraction of true positive helices detected against an increasing threshold of mean false positives per query alignments.Finally, Rivas reappraises the evolutionary evidence for a subset of long non-coding RNAs (lncRNAs) using the new helix-level approach. This evaluation uncovers an artifact that arises when covariation is employed to build an alignment for a hypothetical structure, which is then tested for whether its covariation significantly supports the structure. This helix-level reassessment strengthens the argument against these lncRNAs having a conserved secondary structure.All of these results are important and impactful steps forward for RNA researchers.Minor Points:https://academic.oup.com/bioinformatics/article/22/24/2988/208807), does this metric aid R-scape in predicting new structures, either with cacofold or otherwise?Helix level conservation is a powerful way to build consensus structures  can be reevaluated for their utility?Didn’t proofread extensively but found minor typos, like “mayority” instead of “majority” on page 9 for example.Reviewer #3: The author is looking to extend the work presented in Rscape on statistical identification of evolutionarily conserved RNA structure from the base pair level to the helix level, as RNA base pairs do not occur in isolation.The author explores four statistical methods to aggregate p-values: Fisher, Lancaster, Sidak, and a weighted Fisher approach.The p-value aggregation approach is hypothesized increase sensitivity, and the author proposes a benchmark to assess whether that is associated with a loss of specificity, using real and synthetic RNA datasets, respectively with positive examples and decoys.The author observes that sensitivity/specificity is increased in structured RNAs, offering higher sensitivity with lower false positive rates. As an application, the author looks at potentially conserved structured in well-known long non-coding RNAs, uncovering more precise information than was previously available with state of the art tools.This paper is well structured, with a well defined objective and hypothesis, transparent and logical methods, clearly presented results and relevant exploratory applications. In particular, the construction of the decoys for the sensitivity/specificity benchmarks is very elaborate and strongly contributes to making the results more convincing.My only suggestion would maybe suggest discussing why three of the four methods appear to perform very similarly (and extremely well) whereas sidak seems to underperform a bit, as this result raises some curiosity in a non-expert eye. While this is not necessary for the results presented to be convincing, it may help the discussion.**********Have the authors made all data and (if applicable) computational code underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data and code underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data and code should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data or code —e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********what does this mean?). 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Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript.If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. 5 Jun 2023Attachmentresponse.pdfSubmitted filename: Click here for additional data file. 12 Jun 2023Dear Dr Rivas,We are pleased to inform you that your manuscript 'RNA covariation at helix-level resolution for the identification of evolutionarily conserved RNA structure' has been provisionally accepted for publication in PLOS Computational Biology.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. 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RNA folding is an inherent characteristic of RNA that plays a crucial regulatory function in all stages of post-transcriptional gene expression . This ReRecent developments in both technology and scientific knowledge have significantly promoted the Plant RNA Structure research field. This Research Topic lists two research articles and four mini-reviews with peer review. It aims to summarise the latest advances in plant RNA structure studies, emphasizing the biological significance of RNA structure and outlining potential future research directions.Jaramillo-Mesa et al.; Jin et al.; Sun et al.). Dimethyl sulfate (DMS) is a commonly used chemical that reacts with the Watson-Crick faces of single-stranded Adenine and Cytosine nucleotides . Another chemical-based profiling method is selective 2′-hydroxyl acylation analysed by primer extension (SHAPE) which detects the single-strandedness by acetylating the 2′-OH group of accessible RNA nucleotides. One widely used SHAPE chemical reagent is 2-methylnicotinic acid imidazolide (NAI) . Jin et al used DMS mutational profiling with high-throughput sequencing (DMS-MaPseq) and profiled in vivo RNA secondary structures in rice (Oryza sativa) at both target-specific and genome-wide scales. Their work revealed in vivo RNA structure features of OsPHO2, a key regulator of inorganic phosphate (Pi) homeostasis and the phosphate-starvation response pathway. Notably, the 5’UTR of OsPHO2 contains the OsmiR399 target site, where a single-stranded structure immediately downstream of the miRNA target site was observed, suggesting a regulatory function of mRNA structure on miR399-mediated Pi homeostasis. This demonstration of RNA secondary structure features under the phosphate-starvation condition in rice shows promise for future investigations of RNA structure in rice and other crops in response to nutrition deficiency.Technological breakthroughs in studying RNA secondary and tertiary structures have improved our understanding of RNA structure functionality in plants. For RNA secondary structure profiling, several chemical-based profiling methods were developed in plants . Jaramillo-Mesa et al. used SHAPE to validate the internal ribosomal entry site (IRES) structure in Triticum mosaic virus (TriMV). This guided the design of anti-sense oligonucleotides in repressing viral translation. Moreover, this work showed the role of IRES core modular RNA domains and their potential “free-for-interaction” status in both ribosomal recruitment and start codon selection in a multi-AUG viral 5’UTR. These mechanisms involving structural changes in RNA facilitate translational regulation in plants. The RNA structure profiling techniques used in this study allowed to investigate the secondary structure characteristics of plant viral RNAs, an area where earlier research was predominantly restricted to in silico and in vitro studies. The minireview of Ma and Wang provides an overview on RNA structure studies applied to viroids and how tertiary structural motifs in RNA were systematically identified and shown to have important roles in the mediation of viroid movement, including nuclear import and trafficking across diverse cellular boundaries in plants.SHAPE has been widely used to study RNA structure functionality . The authors also propose potential crop molecular breeding strategies based on the RNA structure-associated regulation of gene expression, such as translation and RNA degradation . In addition, they suggest that riboSNitches, as single nucleotide variants (SNVs) resulting in RNA structure disparities, may offer new ways for interpreting the results of genome-wide association studies (GWAS) . Moreover, riboSNitches may offer potential as molecular markers for breeding line selection to boost future molecular breeding endeavours.The review by Thomas et al.). Notably, most of these environmental conditions influence RNA structure dynamics . The authors comprehensively compared RNA secondary structure thermosensitivities between prokaryotes and eukaryotes, highlighting the role of RNA structure in mediating thermoregulation in plants. The RNA structure motif termed RNA G-quadruplex (RG4) previously known to provide thermotolerance of RNA to cold emerged as a new type of thermosensor regulating plant responses to temperature . In their minireview, Liu et al. summarize technological advances, including both chemical profiling and cell imaging, for determining the RG4 folding status in living cells. The authors also thoroughly compare different methods for RG4 detection and summarize novel functions of RG4s in regulating RNA stability and translation and, thus, plant growth and development at different temperatures. The authors suggest that the evolution of RG4 folds contributed to plant evolution and plant adaptation to the habitats found across the plant kingdom.As sessile organisms, plants are comparatively more prone than other eukaryotes to the effects of environmental conditions such as changes in temperature (The current dynamic and rapid advances in the plant RNA structure field is illustrated by the wide-ranging articles included in this Research Topic. Improving comprehension of the functional significance of RNA structure in various plant biological processes will usher a new era of research on the regulation of gene expression by modifications in RNA structure. By enhancing the research capacity for conducting genome-wide RNA structure studies across extensive plant species and viral RNAs, this progressive understanding will also facilitate the identification of both conserved and divergent RNA structures among natural variants and diverse plant species. Such studies are expected to offer opportunities for investigating the role of RNA structure in plant evolution and habitat selection. Technological advances of RNA structure probing coupled with third generation high-throughput sequencing platforms, such as Pacbio and Nanopore, will achieve high-resolution RNA structure information e.g., at single-molecular and single-cell levels. Further investigations into plant RNA structure dynamics under diverse abiotic and biotic stresses are likely to reveal more RNA structure-mediated regulations in plant responses to environmental changes. Insights into the role of RNA structure in plant thermotolerance and in thermosensing to regulate gene expression has great potential for crop enhancement strategies to mitigate the global impact of climate change on yield. The accelerating study of RNA structures will enable the scientific community to tackle these and other challenges rapidly in upcoming years, offering critical and exciting new insights into our comprehension of RNA structure-mediated regulatory mechanisms in plants.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


N6-methyladenosine (m6A) level was significantly increased after sexual maturity. Functional enrichment analysis of differentially methylated mRNAs screened between the two groups after sequencing showed that they mainly regulated cell development, spermatogenesis, and testicular endocrine function. Functional analysis of the differentially expressed genes screened between the two groups showed that they were involved in the biological processes of mitosis, meiosis, and flagellated sperm motility. Finally, several genes related to spermatogenesis were screened for further verification.The methylation level of testicular tissue in young yaks and adult yaks was determined, and the N6-methyladenosine (m6A) RNA modification has been reported to regulate spermatogenesis and reproductive function in mammals, the molecular mechanism of m6A in yak testis development and spermatogenesis remains largely unknown. Therefore, we collected testicular tissue from juvenile and adult yaks and found that the m6A level significantly increased after sexual maturity in yaks. In MeRIP-seq, 1702 hypermethylated peaks and 724 hypomethylated peaks were identified. The hypermethylated differentially methylated RNAs (DMRs)  mainly regulated spermatogenesis. Functional enrichment analysis showed that DMRs were significantly enriched in the adherens junction, gap junction, and Wnt, PI3K, and mTOR signaling pathways, regulating cell development, spermatogenesis, and testicular endocrine function. The functional analysis of differentially expressed genes showed that they were involved in the biological processes of mitosis, meiosis, and flagellated sperm motility during the sexual maturity of yak testis. We also screened the key regulatory factors of testis development and spermatogenesis by combined analysis, which included BRCA1, CREBBP, STAT3, and SMAD4. This study indexed the m6A characteristics of yak testicles at different developmental stages, providing basic data for further research of m6A modification regulating yak testicular development.Studying the mechanism of spermatogenesis is key to exploring the reproductive characteristics of male yaks. Although  Bos grunniens) are an animal genetic resource known as “the boat of the plateau” and “the omnipotent livestock” in the Tibetan areas of China. They live in high-altitude areas above 3000 m and can tolerate cold and hypoxic conditions [Yaks  is the most abundant internal modification of mRNA and a key mediator of gene expression control from yeast to animals and plants [6A modification can affect transcription, splicing, localization, translation, stability, and post-transcriptional regulation of gene expression at the RNA level [6A regulators maintain dynamic m6A modification [6A methylation, participate in downstream translation and mRNA degradation, and accelerate the speed of mRNA nucleation [6A plays an important regulatory role in cell fate transition and animal development [d plants ,9,10. m6NA level . RNA m6Afication , where mcleation . m6A plaelopment .6A modification is involved in spermatogonial proliferation, spermatogenesis, and reproductive function in mammalian reproduction [6A regulators are expressed in both spermatogenic cells and somatic cells [6A and disrupt the proliferation of spermatogonial stem cells, hindering spermatogenesis. YTHDC2 is essential for the gene expression program of the male germline from mitosis to meiosis [6A-seq and RNA-seq on porcine spermatogonia, spermatocytes, and round sperm cells and analyzed the mechanism of m6A in spermatogenesis. Wang et al. [6A modification sites of yak and cattle–yak testis and discussed the effect of m6A on spermatogenesis arrest of cattle–yak. Liu et al. [6A in bovine testis at different developmental stages by MeRIP-seq analysis and identified several key regulatory genes related to the regulation of mammalian testis development and spermatogenesis.In particular, moduction ,16,17. Sic cells . ALKBH5 ic cells ,20. ALKBic cells . Lin et ic cells  showed t meiosis ,24, and  meiosis . Liu et  meiosis  performeg et al.  comparedu et al.  analyzed6A regulates bovine spermatogenesis has been reported, the impact of m6A in yak testis development and spermatogenesis remains largely elusive. To further investigate the regulatory role of m6A on spermatogenesis in yak testis, we collected 6-month-old and 30-month-old yak testicular tissue samples for MeRIP-seq. This allowed us to obtain the m6A spectrum of the transcriptome at different developmental stages of yak testis. Our study provides a theoretical basis for the role of m6A during spermatogenesis in testis development after birth, as well as preliminary data for the research of the yak reproduction mechanism essential to yak breeding.Although the mechanism by which mAll animal procedures were performed according to the China Council on Animal Care guidelines and the Ministry of Agriculture of the People’s Republic of China. The yak handling procedures were approved by the Animal Care and Use Committee of the Lanzhou Institute of Husbandry and Pharmaceutical Sciences Chinese Academy of Agricultural Sciences (Permit No: SYXK-2014-0002).3, which was fixed in 10% neutral formaldehyde for producing tissue sections, and the consistency of the collection site was maintained. The remaining tissue was cut into three parts and put into a clean cryopreservation tube for liquid nitrogen cryopreservation.Six healthy male yaks from Xiahe County, Gannan Tibetan Autonomous Prefecture , were selected. According to the degree of sexual maturity, three 6-month-old yaks (Y6M) before sexual maturity were selected for the first group, and three 30-month-old yaks (Y30M) were selected for the second group; the samples were collected from yaks during the estrus period in August. After local disinfection of the testes, they were castrated by veterinarians to obtain testicular tissue. The collected testicular tissue was washed clean with normal saline, and a scalpel was used to draw along the longitudinal axis of the testis. The testicular parenchyma vertical with the longitudinal axis was collected, and the cut tissue was trimmed into a tissue block with a volume of about 1 cm6A and m6A-related enzymes in the total RNA of testicular tissue were determined . In the transcriptome sequencing analysis, the fragments per kilobase of transcript per million mapped reads (FPKM) value of the gene was used to represent the expression level of the gene. The screening condition for differentially expressed genes between samples was . The condition for screening significantly enriched items in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was p < 0.05. Detailed experimental steps and data analysis process are shown in The steps of RNA extraction and quality control, library construction, sequencing, and bioinformatics analysis refer to the methods reported by Guo et al. . The levtermined . QualifiBRCA1, CCNE2, SOX5, PDE4A, INHBA, ERBB4, IGFBP4, and SMAD4 were selected to verify the sequencing results by qRT-PCR. Data were expressed as mean ± standard error (n = 3). The glyceraldehyde-3-phosphate dehydrogenase gene was used as an internal reference gene, and the 2−ΔΔCT calculation method was used to standardize mRNA expression [p ≤ 0.05 was considered statistically significant. The primer synthesis of the target gene was performed using the NCBI website  and sexual maturity stage  are shown in 6A plays a regulatory role in yak testis development, the m6A levels of testicular tissue before and after sexual maturity were first compared. The m6A level of yak testicular tissue after sexual maturity was significantly (p < 0.05) higher than that at the juvenile stage . The expression of FTO and ALKBH5 was significantly (p < 0.01) higher in sexually mature yak testis than in juvenile yak testis (p < 0.01) increased  difference in yak testis before and after sexual maturity  were identified in Y6M and Y30M samples   , of whic| > 1.5) c,d. The | > 1.5) f. In the| > 1.5) g. Enrich6A modification and gene expression, a combined analysis of m6A-seq and RNA-seq data was performed . Among the 1013 m6A hypermethylated peaks, 581 mRNAs were up-regulated, and 432 genes were down-regulated. Among the 463 m6A hypomethylated peaks, 170 genes were up-regulated, and 293 genes were down-regulated  by tightly connecting the adherens junction and gap junction to prevent the immune system from attacking its own germ cells [We found several mrm cells . Simultarm cells . Destrucrm cells . The Wntrm cells ,72. In pEFCAB2 gene was significantly up-regulated in the testis of sexually mature yaks. We also found that the WDR5 gene is one of the up-regulated DEGs. WDR protein plays an important role in spermatocyte division, sperm flagellar assembly, and head formation [The transcriptome results showed that DEGs screened after sexual maturity were closely related to calcium ion binding, spermatogenesis, cell differentiation, cilium assembly, and flagellated sperm motility and were significantly enriched in ECM–receptor interaction, cortisol synthesis and secretion, and aldosterone synthesis and secretion pathways. In mammals, cilia are essential for development, sensation, cell signaling, and sperm motility. Calcium-binding proteins regulate signaling pathways necessary for ion metabolism and sperm maturation events. Shawki et al. identified EFCAB2 as a new calcium-binding protein in mouse testis, which may regulate sperm flagellar movement . In our ormation . ECM incormation . Cortisoormation . Overall6A-seq and RNA-seq showed that m6A methylation was specifically expressed in yak testicles at different developmental stages, and these regulatory factors were crucial for maintaining spermatogenesis. BRCA1 is a nuclear phosphoprotein expressed in a broad spectrum of tissues during cell division [BRCA1 is expressed in the spermatogonia and premeiotic spermatids in the testis [CREBBP is expressed in Sertoli cells and undifferentiated spermatogonia and may participate in regulating the activation of the follicle-stimulating hormone-mediated transferrin promoter in the testis by connecting bHLH and CREB activities [STAT3 is widely expressed in the supporting cells of the entire seminiferous tubules, and the expression level of STAT3 in the seminiferous tubules is closely related to spermatogenesis [SMAD4 is expressed in mammalian testis and plays an important role in testis development and spermatogenesis [6A methylation affects testis development and spermatogenesis by regulating the translation and degradation of BRCA1, CREBBP, STAT3, and SMAD4.Combined analysis results of mdivision . BRCA1 ie testis , and BRCe testis . CREBBP tivities . CREBBP tivities . STAT3 itivities . In mousogenesis . When STogenesis . SMAD4 pogenesis . Our res6A modifications occurred during the sexual maturity of yak testis, and the m6A level of yak testicular tissue was significantly higher after sexual maturity than at the juvenile stage. By measuring the expression of methylation-related enzymes, we found that yak testis development and spermatogenesis were mainly regulated by demethylases. The differential expression of m6A-methylated mRNAs involved multiple biological pathways, such as Wnt, PI3K, and mTOR signaling pathways, and participated in germ cell proliferation, cell differentiation, spermatogenesis, sperm motility, and other processes. Finally, we also screened the key regulatory factors affecting testis development. Subsequently, the screened key regulatory factors will be verified at the cellular level, and the molecular mechanism of these regulatory factors regulating spermatogenesis through m6A will be further explored. This study provides new insights into further understanding the epigenetic molecular regulation mechanism of testicular development.In summary, we found that a large number of m