PPR (pentatricopeptide repeat), OZ1 (organelle zinc-finger), and MORF/RIP gene expression levels in wheat were also examined. Normal growth conditions diverge gene expression of these three different gene families, implying that normal growth conditions for various genotypes can modify RNA editing events and have an impact on gene expression levels. While the expression of PPR genes was not change. We used Variant Effect Predictor (VEP) to annotate RNA editing sites, and Local White had the highest RESs in the CDS region of the protein. These findings will be useful for prediction of RESs in other crops and will be helpful in drought tolerance development in wheat.Wheat is an important cereal and half of the world population consumed it. Wheat faces environmental stresses and different techniques  were used to enhance its production but RNA editing (RESs) is not fully explored in wheat. RNA editing has a special role in controlling environmental stresses. The genome-wide identification and functional characterization of RESs in different types of wheat genotypes was done. We employed six wheat genotypes by RNA-seq analyses to achieve RESs. The findings revealed that RNA editing events occurred on all chromosomes equally. RNA editing sites were distributed randomly and 10–12 types of RESs were detected in wheat genotypes. Higher number of RESs were detected in drought-tolerant genotypes. A-to-I RNA editing  sites were also identified in six wheat genotypes. Most of the genes were found to be engaged in molecular processes after a Gene Ontology analysis.  RNA editing is a fundamental factor in primate evolution, and it plays a crucial function in regulating environmental stress. Previous study has revealed the wide range of adenosine (A) to inosine (I) editing in mammalian transcriptomes that are involved in biological processes such as nervous system development [RNA editing was discovered 30 years ago. RNA editing is a post-transcriptional variation of transcripts encoded by the chloroplast, nucleus, or mitochondrial genome of animals and plants, and it was first reported in protozoan mitochondria and then in plant –4. RNA melopment , 14.The identification of RNA editing targets is an important first step in gaining a deeper understanding of this post-transcriptional modification and entire genome and transcriptome sequence of the same individual to exclude polymorphisms and mutations between populations, including experimental methods with the necessary high-throughput sequencing and background resolution are required , 16. DeeTriticum aestivum L.) is a major staple food crop grown in both irrigated and rainfed locations around the world, accounting for 17 percent of arable land and provides important food calories, especially to the 4.5 billion people who live in poor countries [OZ1 and MORF/RIP or RNA editing factor stability. Drought promotes fast RNA editing of the wheat transcriptome, resulting in a shift in the ratio of edited to unedited proteins in the proteome. According to our findings, amino acid alterations in these genes may also play a role in wheat stress adaptation. These key findings tell the story that genetic variation is common, highlighting the significance of a thorough description of these polymorphisms for better understanding wheat growth under normal conditions.Wheat (ountries –22. Wheaountries . The scoThe seeds of drought-sensitive wheat (Batis and Blue Silver) and drought-tolerant wheat , were germinated on moist germination paper. After 72 h of germination, four germinated seeds of each genotype were shifted in soiled filled pots consisting of PVC pipes (4.5 cm diameter × 45 cm depth). Each genotype was repeated thrice. Pots were kept at 24/12 ± 2°C Day/night temperature with 10 hours photoperiod in the field. The seedlings were grown under well-watered condition for 35 days. The whole experiment was conducted during wheat growing season in natural conditions. At 35-days-old-seedling stage of wheat, roots samples (each data point pooled from eight plants) were taken as previously described , 28 for TRIzol reagent kit  was used to extract total RNA from root tissues of wheat cultivars. The NanoDropTM 1000 spectrophotometer (Thermo Scientific®) was used to determine the quality and amount of RNA. RNA samples were sequenced from commercial company . Subsequently, paired end (PE) RNA-Seq libraries were prepared, and RNA sequencing was performed using Illumina NGS HiSeq2000 platform. Number of reads obtained per lane was significantly high, ranging from 10.5 to 13.3 million per sample with average reads per sample was around 12 million. Reads were 100 nt in length. Trimmomatic-0.33  was usedhttps://sprint.tianlab.cn/, SnP-free RNA editing Identification Toolkit) by using default setting [ftp://ftp.ensemblgenomes.org/pub/release-25/plants/fasta/triticum_aestivum/dna/). The overlapped data from both methods were kept for further investigation. The editing sites were then further filtered using the following criteria: (1) editing reads to total mapped reads ratio greater than 50; (2) edited sites with more than five mapped reads. The Ensemble Variant Effect Predictor (VEP) tool (https://asia.ensembl.org/info/docs/tools/vep/index.html) with default parameters was used to annotate the RNA editing site [All potential RES were calculated with the help of SPRINT , OZ1 (organelle zinc-finger), and RIP (RNA editing factor interacting proteins) gene families in wheat were assessed and compared using NOIseq package. The protein sequences of Triticum aestivum PPR, OZ1, and Multiple organellar RNA editing factor (MORF)/ RIP were obtained from the Ensembl database [http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/) was used for quality checking and Trimmomatic tool was also used for trimming of reads . NOIseq package [Using transcriptome analysis of RNA-seq data, the levels of gene expression of the database . The prodatabase . In deta package  was used package .PPR, RIP, and OZ1 gene families in roots of wheat under the same condition used for RNA-Seq analysis. qRT-PCR studies were done using a Step One Plus Real-Time PCR System  using SYBR Premix Ex Taq  and a Prime Script RT reagent Kit with gDNA Eraser . To standardize the expression data, the wheat gene RLI (RNase L inhibitor-like protein) was used as an internal control [qRT-PCR tests were used to examine the expression of the  control . The 2-Δ control , 40. ThrTotal RNA from roots was collected and sequenced using the Illumina HiSeq 2500 platform to gain a better understanding of drought-sensitive and drought-tolerant genotypes. The quality control of raw reads was checked to detect sequencing errors by analyzing sequence quality, GC percentage, the adaptors sequences, and duplicated reads . The accThe levels of stringency employed to reduce false positives affect the computational detection of RNA editing by RNA seq. This is especially true when using transcriptome data only . Only RNTraesCS1D02G346400 has maximum 21 RESs and RNA-editing sites were discovered utilizing the methods mentioned above [TraesCS1A02G353700 and TraesCS2D02G564600 have maximum 19 RESs. There were 30 RESs detected on 7 non protein coding genes  and lowest transversion rate with number of  followed by Local White with the number of transition  and transversion , UZ-11-CWA-8 with the number of transition  and transversion , Batis with the number of transition  and transversion , Blue Silver with the number of transition  and transversion  and Chakwal 50 with the number of transition  and transversion , these results are consistent with the previous studies [PPR, MORF/RIP, and OZ1) families which might be involved in RNA editing process. In wheat, a total of 24, 29, and 278 proteins were identified as OZ1, MORF/RIP and PPR, respectively, by using key words in wheat Ensembl Plants database (https://plants.ensembl.org/Triticum_aestivum/Info/Index). The expression level of these gene families in different type of wheat genotypes were measured by RNA seq. The expression level of most of OZ1 gene family is higher in Local White, Chakwal 50, UZ-11-CWA-8, and Syn-22 in which higher number of RESs were identified, implying that normal growth conditions for different genotypes might modify RNA editing events and have an impact on gene expression levels in these drought-tolerant genotypes. Expression levels of this gene family by RNA seq in different genotypes of wheat also shown in OZ1 gene family also determined by qRT-PCR. The expression level of these genes TraesCS3A02G396300 (OZ1), TraesCS2A02G226400 (OZ2), TraesCS3B02G225200 (OZ3), and TraesCS3B02G370800 (OZ4) by qRT-PCR is shown in To figure out why six wheat genotypes had varying numbers of RNA editing? We looked at the expression of three gene (MORF/RIP to see why six wheat genotypes had varying numbers of RNA editing? More genes of this family were upregulated in drought-tolerant genotypes identified by RNA-seq data as shown in TraesCS5B02G290000 not expressed in any genotype of wheat. The expression level of 4 genes TraesCS6D02G007100 (RIP1), TraesCS4D02G104800 (RIP2), TraesCS7A02G050200 (RIP3), and TraesCS5B02G289900 (RIP4) of this family also noticed by qRT-PCR as shown in We also looked at the expression of PPR genes in six wheat genotypes were measured and compared using transcriptome analysis of RNA-seq data. However, no significant differences in expression were found for PPR genes under normal growth conditions, implying that normal growth conditions solely affect RNA editing events and had no effect on expression levels for this gene family as compared to OZ1 and RIP gene families. Surprisingly, only six PPR genes showed a high level of expression across all genotypes, as shown in TraesCS6B02G249200 (PPR1), TraesCS6D02G202700 (PPR2), TraesCS5B02G038400 (PPR3), and TraesCS5A02G037400 (PPR4) from this gene family were determined by qRT-PCR  term also find and mostly genes involved in nitrogen compound metabolic process as shown in To determine the functions of the genes in which RNA editing site was detected in this study, all REGs in each genotype were annotated to the terms in the GO database. Results showed that 26, 23, 21, 18, 26, and 20 significantly enriched molecular functions identified in Batis, Blue Silver, Chakwal 50, Local White, Syn-22, and UZ-11-CWA-8, respectively. The most enriched terms identified in each genotype are as energy derivation by oxidation of organic compounds, nucleosome organization, chromatin assembly, DNA packaging, aerobic electron transport chain, DNA conformation change, protein-DNA complex subunit organization, ATP synthesis coupled electron transport, oxidative phosphorylation, electron transport chain, ATP metabolic process, amide biosynthetic process, response to water deprivation, protein-DNA complex assembly, respiratory electron transport chain, cellular amide metabolic process, heat shock protein binding, response to water, peptide metabolic process, response to inorganic substance and peptide biosynthetic process and proline dehydrogenase activity as shown in RNA editing is the epigenetic machinery that causes deletions, insertions, and substitutions in transcripts, as well as the increase of the transcriptome’s complexity through the diversification of genomically encoded information. It was reported in Kinetoplastid protozoa and many species . RNA ediRNA-seq is a powerful technique for identifying all possible RNA editing sites and quantifying their editing extent, particularly for sites with low editing extent. As a result, with the introduction of sequencing technology, RNA sequencing is now utilized to find RESs in numerous organisms –59. The PPR gene family has been identified in many plant species such as Arabidopsis thaliana and Oryza sativa, and involved in RNA processing [OZ1 binds to RIP protein and interact with PPR [OZ1 and RIP is also higher in drought-tolerant genotypes. This makes them more adaptive to environmental stress. Despite this, one PPR member in any genotypes of wheat was not expressed, this gene may have specific role in drought-tolerant genotypes. Further effort needs to be put into clarify their involvement (or not) in RNA editing.This conclusion is in line with previous research that demonstrated RESs controls plant development, organ formation, growth in the stress condition , 70. In ocessing , 72. Accwith PPR . Our resOZ1, PPR1, and RIP gene families in drought-tolerant and susceptible genotypes. More RESs were detected in drought-tolerant as compare to drought-susceptible genotypes. RNA editing not only controls plant organ formation and development but also plays an indispensable role in response to diverse environment conditions and suggesting that RNA editing might act as a “regulator” to control the root architecture development. Furthermore, the regulatory network of the essential genes in which RESs were detected were investigated. Nitrogen compound metabolic process and phytohormone signal transduction might play important role in roots development which helps wheat plant adaptation to harsh climatic conditions. These findings will contribute to improve understanding the molecular mechanism of dynamics of gene regulation in root architecture development in wheat at posttranscriptional level.In this study, we systematically identified the RNA editing sites (RESs) in roots tissue of wheat and checked the gene expression analysis of S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 File(XLSX)Click here for additional data file.S4 File(XLSX)Click here for additional data file.S5 File(XLSX)Click here for additional data file.S6 File(XLSX)Click here for additional data file.S7 File(XLSX)Click here for additional data file.S8 File(XLSX)Click here for additional data file.S9 File(XLSX)Click here for additional data file.S10 File(XLSX)Click here for additional data file.S11 File(XLSX)Click here for additional data file.S12 File(XLSX)Click here for additional data file.S13 File(XLSX)Click here for additional data file.S14 File(XLSX)Click here for additional data file.S15 File(XLSX)Click here for additional data file.


Cancer metastasis occurs due to several causes such as histologic, genetic, and pathologic features. Hematogenous spread, lymphatic spread, and direct spread to pleura are the main routes of lung metastasis . The priMany interesting theories exist regarding the origin of metastatic cells. Jamil and Kasi summarized that there would be main roots for the lung metastasis among these theories: 1). epithelial-mesenchymal transition (EMT) in which epithelial stem cells transform into mesenchymal cells, 2). stem cell origin of metastatic tumors in which tissue stem cells are the origin of metastatic cancers, 3). a concept of macrophage facilitation of metastasis in which tumor-associated macrophages contribute to tumor progression, and 4). myeloid cell origin of metastasis in which myeloid origin cells with mesenchymal properties promotes metastasis . The sigvia JAK2/STAT3/miR-506-3p/FoxQ1 axis activated by IL-6 (Jamil and Kasi highlighted the importance of tumor-derived exosomes containing proteins, DNA, RNA, and non-coding RNA. Tumor-derived exosomes may prepare the pre-metastatic niche, and establish a microenvironment at distant sites . Tumor-d by IL-6 .Systematic symptoms of patients with lung metastasis are fatigue, nausea, anorexia, and weight loss, while localized symptoms include pleurisy/pleural effusion, cough, dyspnea, hemoptysis, scalp metastasis, electrolyte disturbances, Pancoast tumor, and superior vena cava syndrome . SeveralThe prognosis of lung metastasis varies depending on the type of tumor, molecular biomarkers, extent of the disease, or treatment modalities . Complic+ tumor-infiltrating lymphocytes, which are important factors of cancer progression, are linked via miR-200 and ZEB1 transcription factor (via inhibition of miR-33a-5p and activation of Wnt/β-catenin signaling (via STAT-3 induced EMT, which may be a candidate biomarker for cancer metastasis (The understanding of lung metastasis with the view of EMT mechanism and RNAs would lead to the comprehensive therapy of cancers. Several kinds of RNAs including microRNAs are involved in tumor-derived exosome-mediated lung metastasis. EMT and immunosuppression of CD8n factor . The miRignaling . A paneltastasis . A multitastasis . EMT mectastasis . Furthertastasis . Regulattastasis . The pot


Therefore, applications of deep sequencing to RNA face the challenge of how to efficiently enrich mRNA species prior to library construction. Traditional methods extract mRNAs using oligo-dT primers targeting the poly-A tail on mRNAs; however, this approach is not comprehensive as it does not capture mRNAs lacking the poly-A tail or other long non-coding RNAs that we may be interested in. Alternative mRNA enrichment methods deplete rRNAs, but such approaches require species-specific probes and the commercially available kits are costly and have only been developed for a limited number of model organisms. Here, we describe a quick, cost-effective method for depleting rRNAs using custom-designed oligos, using chickens as an example species for probe design. With this optimized protocol, we have not only removed the rRNAs from total RNAs for RNA-seq library construction but also depleted rRNA fragments from ribosome-protected fragments for ribosome profiling. Currently, this is the only rRNA depletion-based method for avian species; this method thus provides a valuable resource for both the scientific community and the poultry industry.Deep sequencing of RNAs has greatly aided the study of the transcriptome, enabling comprehensive gene expression profiling and the identification of novel transcripts. While messenger RNAs ( RNA-seq) or ribosome protected fragments (Ribo-seq) has greatly aided in the characterization of the transcriptome, enabling gene expression studies and the identification of novel transcripts and novel open reading frames (The high-throughput sequencing of cellular RNAs (g frames . ProteinrRNAs). Because ribosomes are required for protein synthesis in the cytoplasm and mitochondria, rRNAs are abundant, accounting for ~80% of total RNA in eukaryotes  end processing . An altekaryotes . One of karyotes , transcrRPFs) on a genome-wide scale, with a sub-codon resolution  are currently the only type of PGCs that can be cultured in vitro for long term  and females are the heterogametic sex (ZW). Chicken primordial germ cells .The RNA depletion protocol outlined here consists of 2 parts: the oligo-based rRNA depletion  and the To design the antisense oligo, we downloaded the rRNA sequences of both cytosolic ribosomes and mitochondrial ribosomes in chicken species from NCBI. These are mitochondrial rRNAs including the 16S rRNA (XR_003076910.1), and 12S rRNA (MH879470), and cytosolic rRNAs which for eukaryotes include 18S rRNA (AF173612.1), 28S rRNA (XR_003078040.1), 5.8S rRNA (XR_003078039.1) and 5S rRNA (XR_003076910.1). In order to minimize the chances of mismatch, for species not close related to chicken, a specific set of oligos should be designed and synthesized according the following steps.https://github.com/LiLabZhaohua/rRNADepletion].•BLAST) was used to find possible pairing with other RNAs in chickens. We confirmed that these oligos do not have significant targets in the chicken transcriptome , by mixing the 5.8S, 28S, 18S, 16S, 12S, and 5S oligos to a final concentration of 0.5 μM for each oligo.DNA oligos were designed by converting the original sequences to their reverse complements and splitting the full-length sequences into 50 nt non-overlapping windows using a python script [This step is done with the mirVana total miRNA isolation kit , but any other total RNA isolation kits can be applied to this step.•Remove the tissue from the −80°C freezer.•Cut out 25 mg of frozen tissue and place into a 1.7 uL tube.•Add 300 uL Lysis/Binding Buffer into a plastic weigh boat or tube on ice.•Grind frozen tissue into a powder using a homogenizer.•Add 30 uL miRNA Homogenate Additive to a tube. (Note: It is important to add the Homogenate additive immediately to the tube after the tissue is ground as it prevents the degradation of RNA.)•Leave mixture on a rotator for 10 min.•Vortex the mixture for 60 s to mix well.•Centrifuge the mixture for 5 min at 16,900 RCF at room temperature. •Transfer the upper layer to a new 1.7 uL tube. It should be approximately 300 uL. It is very important to not disturb the bottom phase since it is mostly tissue and acid phenol.Add 300 uL of acid chloroform (Thermo Fisher cat. AM9722). Withdraw the bottom layer, avoiding the upper phase as this is mostly aqueous buffer.•Add 375 uL of room temperature 100% ethanol to the aqueous phase.•Place the column on a collection tube.•Pipet the mixture to the column.•Centrifuge for 30 s at 9,300 RCF.•Discard the flow through.•Add 700 uL miRNA wash solution 1 (Premixed with ethanol).•Centrifuge for 10 s.•Discard the flow through.•Add 500 uL miRNA wash solution 2/3 (Premixed with ethanol).•Centrifuge for 10 s.•Discard the flow through.•Add 500 uL miRNA wash solution 2/3 (premixed with ethanol).•Centrifuge for 10 s.•Discard the flow through.•Centrifuge for another 3 min to remove the liquid residual.•Preheat the nuclease-free water to 95°C.•Transfer the column into a new collection tube.•Add 100 uL preheated elution buffer or nuclease-free water.•Spin for 4 min at 11,300 RCF at room temperature.•Collect the elutes and store it at −80 °C.Wash solution 1 and solution 2/3 should be pre-mixed with ethanol according to the manual. At the end of the final stage, RNA can be eluted with either nuclease-free water or in the Elution buffer provided by the kit.For this step, the enrichment for mRNAs and RPFs follows the same steps except for the temperature for RNase H treatment and the last step, in which the depletion from total RNAs uses the RNA Clean & Concentrator kit  protocol, while the depletion from RPF uses the acid phenol protocol.In this step, we prepare 3 solutions that will be used for rRNA depletion later.For the probe oligo, prepare the 5X rRNA oligo hybridization buffer and 10X RNase H and aliquot for use.Prepare 5X rRNA oligo hybridization buffer: mix 100 mM Tris-HCl with 200 mM NaCl.2.Prepare 10X RNase H digestion buffer: mix 500 mM Tris-HCl with 1 M NaCl and 200 mM MgCl•Pipette 2 μg of RNA and 15 μL DNA oligo probe into a tube, adjusting the volume of 5X rRNA oligo hybridization buffer and water according to •You may adjust the volume and choose one system in •You may also dilute the system, using the 1X rRNA oligo Hybridization Buffer, after you mix all the components (not over 45 μL for 2μg RNA).•Use the PCR machine for annealing: Heat to 95°C for 2 min and slowly cool down (−0.1 °C/sec) to 22°C, then incubate at 22°C for additional 5 min.•Turn on the PCR machine and set the temperature to 45°C.•Add the solutions and Invitrogen RNase H (Thermo Fisher cat. 18021071) listed in •Incubate at 45°C for 1 h in the PCR machine.•Take out samples from the PCR machine and adjust the PCR machine to 37°C.•Immediately add the components listed in •Incubate at 37°C for 30 min in the PCR machine.•Transfer 50 uL of RNA to a new 1.7 mL tube.•Add 50 uL water to the tube.•Add 200 uL adjusted binding buffer to the tube.•Transfer the mixture into a column.•Centrifuge for 30 s at 16,900 RCF at room temperature.•Discard the flow-through.•Add 200 uL RNA prewash.•Centrifuge for 30 s at 16,900 RCF at room temperature.•Discard the flow-through.•Add 200 uL RNA wash buffer.•Centrifuge for 30 s at 16,900 RCF at room temperature.•Discard the flow-through.•Add 200 uL RNA wash buffer.•Centrifuge for 2 min at 16,900 RCF at room temperature.•Discard the flow-through.•Transfer the columns into new 1.5 mL tubes (RNase Free tube).•Add 15 uL DNase/RNase-free water directly to the column matrix.•Let the columns sit at room temperature for at least 1 min.•Centrifuge for 30 s at 16,900 RCF at room temperature.•Keep the flow through in the tube and store the tube at −80°C immediately.This step uses the RNA Clean & Concentrator kit (Thermo Fisher cat. R1013) protocol. Prepare adjusted RNA Binding buffer by mixing equal volumes of buffer and ethanol.•Pipette 2 μg of RNA and 15 μL DNA oligo probe into a 1.7 mL tube.•Adjust the volume of 5X rRNA oligo hybridization buffer and water according to •You may adjust the volume and choose one system in •You may also dilute the system, using the 1X rRNA oligo hybridization buffer, after you mix all the components (not over 45 μL for 2μg RNA).•Use the PCR machine rRNA program for annealing: Heat to 95°C for 2 min and slowly cool down (−0.1°C/sec) to 22°C, then incubate at 22°C for additional 5 min.•Turn on the PCR machine and set the temperature as 37 °C for RPF for optimal results. * We have compared the treatment temperature at room temperature, 30°C, 37°C, 42°C, and 45°C, and found that 37 °C has the best removal based on our sequencing results .•Add the solutions and RNase H (Thermo Fisher cat. 18021071) listed in •Incubate at 37°C for 1 h on PCR machine.•Remove the samples from PCR machine and adjust the PCR machine to 37°C.•Immediately add the components listed in •Incubate at 37°C for 30 min in the PCR machine.•Transfer 50 uL RNA to a 1.7 mL tube.•2O.Add 200 μL ddH•Add 300 μL acid phenol chloroform (Thermo Fisher cat. AM9722). Note the total volume should be around 550 uL.•Vortex for 1 min to mix well.•Spin at 20,100 RCF for 15 min at room temperature.•Withdraw the upper phase (around 250 uL) without disturbing the lower phase and transfer the solution to a new 1.7 mL tube.•Add 1 μL glycogen .•Add 3 volumes (around 750 uL) of 100% ethanol.•Leave at 4°C for an hour, or −20°C overnight. White precipitate should be visible.•Spin down at 16,900 RCF for 30 min at 4°C.•Wash with 200 uL of 70% ethanol.•Centrifuge at 16,900 RCF for 5 min at highest speed at 4°C.•Pour out the liquid.•Centrifuge at 16,900 RCF for another 3 min at highest speed at 4°C.•Pipette out all the liquid and leave only the pellet in the tube.•Dissolve in water at room temperature.•Store at −80°C https://github.com/LiLabZhaohua/RiboSeqPipeline). Before genome mapping, this pipeline automatically maps Ribo-seq reads to 5S and 45S ribosomal RNA and count the rRNA reads. The rRNA percentage was calculated by dividing rRNA mapping reads to total genome mapping reads. A Student's t test was performed between our method with another method in R 3.5.0 using t test function, with alternative = "less" parameter. 12S rRNA depletion efficiency was measured by RT-qPCR and analyzed using DART-PCR  and thermostable RNase H  from NEB, and RNase (Thermo Fisher cat. 18021071) from Invitrogen with 1 uL probe, 1 uL RNase H treated for 30 min at 37°C and 45°C. The smallest Relative quality value with respect to the control value for RNase H from Invitrogen indicates the best result at 37°C. Then we compared the Invitrogen RNase H and NEB RNase H with 1 uL and 2 uL at 37°C and 45°C with 3 μL probe for 1 h of RNase treatment and then 30 min of DNase treatment. The result indicates that Invitrogen has the best efficiency at 45°C. After the optimal enzyme chosen, the optimal amount of probe was also investigated with 1μL Invitrogen RNase Treatment at 45°C for 1 h and then DNase treatment for 30 min. Different ratios of the RNA and probe, including 1:0, 1:1, 1:2, and 1:3 were tested. In the end, the optimal condition for RNA depletion is 1 μg RNA: 3 μL probe, 0.2 μL Invitrogen RNase H treatment at 45°C for 1 h, and DNase treatment at 37°C for 30 min. The result was expressed as the ratio of 28S to GAPDH, decreased >4,700 fold from 706 to 0.15 after rRNA depletion, measured by RT-qPCR.The 260 of 230 values for "pure" nucleic acid are often used to examine whether contamination such as salt or protein exists in the sample or to measure the purity of nucleic acid before and after. Expected 260 of 230 values are commonly in the range of 2.0 to 2.2. If the ratio is appreciably lower, it may indicate the presence of contaminants which absorb at 230 nm. The ratio would be decrease largely due loss of rRNA. As shown in The 260 of 280 values indicate the purity of RNA. A ratio of ~2.0 is generally accepted as "pure,” while pure DNA has a ratio close to 1.8. As expected, we observed a significant decrease of RNA amount after depletion, as 90% of the RNA, which is ribosomal RNA, is now depleted. The decrease of the RNA concentration indicates that RIN) is an index for assigning integrity values to RNA measurements. No significant change should be observed for a successful experiment before and after DNase treatment. The success of rRNA depletion is also demonstrated by the bioanalyzer results. The RNA integrity number . Our method also outperforms poly-A selection method, which has an average rRNA percentage of 2.1% (Student's t test P = 0.02). We also compared the depletion performance on mitochondria rRNAs. Using another internal control, ACTB, our method has an average 22.67 times decrease of 12S comparing to the untreated sample, which is slightly better than the poly-A selection method, with an average of 19.11 times decrease (Student's t test P = 0.22)  as a comparison to the poly-A selection results from published papers ( = 0.22) .t test P = 0.02), we also found the chicken RNA-seq signals evenly distribute throughout the mRNAs as shown by the metagene analysis of a chicken RNA-seq results , zebra finch (Taeniopygia guttata), north island brown kiwi (Apteryx mantelli) and turkey . As shown in Aves species. Depending on how the oligos are designed and which type of sequence the oligos are specified to target, the same method can potentially be applied for a wide range of organisms and different types of RNA. As long as the oligo is designed to antisense to the specific type of RNA of a specific species, this method should guarantee good quality of purification.Furthermore, we aligned the 18S rRNA sequences from four avian species, including chicken (


Pempheris (Pempheridae) are one of the most abundant nocturnal fishes on coral reefs.Although the biomass of the nocturnal fishes is almost same as that of diurnal fishes, most of the ecological studies that examine feeding or reproductive behaviors are on diurnal fishes. Therefore, there is limited ecological information regarding the nocturnal fishes. This fact may be attributed to the difficulty in observing them during darkness. Members of the genus Pempheris schwenkii were observed by attaching a chemical luminescent tag. Tagged fishes were followed by an observer without torch and SCUBA, and their positions and estimated depths were plotted on an underwater topographic map. Aquarium tank observation was carried out to further describe their habits during the night.The nighttime migrations of The new tagging method provided good data for observing the migration behavior. In all five observations, the target fishes started nighttime migration from the entrance of their cave within 1 h after sunset. All of them immediately left the inner reef and spent most of the observation time near the surface (0–5 m depth) or shallow (5–15 m depth) water-columns of the outer reef. Their migration pattern varied between days, but they migrated long distance (379–786 m/h) during each observation. The behavior observed in the aquarium tank was categorized into five patterns: schooling, shaking, migrating, spawning, and feeding. Shaking and spawning were observed during one of three observation days.P. schwenkii in nature by a new method using chemical luminescent tags. In addition, combined observations from nature and an aquarium could be used to estimate the behavior of this species. Pempheris schwenkii may reduce their predation risk of eggs and adults by spawning at outer reef in nighttime. It was estimated that they can potentially migrate 4–7 km/night. The rapid growth known for this species may have been supported by their feeding behavior where they can fill up their stomach every night with rich zooplankton in outer reefs. Furthermore, the behavior of this species indicates the possibility that they make an important contribution to the flow of energy and materials in their coral reef ecosystem.The present study firstly clarified the small-scale but dynamic nocturnal migration pattern of  In general, both groups hide in their respective shelters during their inactive periods. Pempheris, sweepers, comprise one of the most abundant nocturnal groups of fish in rocky and coral reefs. Characteristically, the fishes of this genus hide in underwater caves, rock recesses, or crevices during the day, and swim out to open water at night, where they primarily prey on zooplankton  on the western coast of Okinawa-jima Island in the Ryukyu Archipelago. The length, width, and depth of the huge half-underwater cave were up to ca. 50 m, 10 m, and 5 m, respectively. Sunlight comes in from outside at the entrance of the cave, and the water and the walls in the cave reflect a blue light. This cave is called “Ao-no-dokutsu”, which means “blue cave” in Japanese, and is one of the most popular diving spots in Okinawa. It contains a number of rock recesses, and several hundred to more than a thousand (based on the seasons) of P. schwenkii inhabit this cave. Approximately 50 individuals of P. adusta and one or two individuals of P. oualensis were observed near the entrance of the cave.The nighttime migrations of A general underwater topographic map of the study area was prepared by combining depth data from a depth finder  and lat/long data from a GPS (Garmin eTrex Venture HC). Directions in the present study were shown from 0–360°, with 0° and 180° meaning north and south, respectively. The coastline of the study area ran straight from northwest (310°) to southeast (130°). The site contains a narrow inner reef (<5 m depth) and grows rapidly deeper (up to 20 m) at the outer reef. The border between inner and outer reefs ran parallel 40–50 m from the coastline. The depth was slightly deeper to the offshore in outer reef by up to 30–40 m.https://eco.mtk.nao.ac.jp/koyomi/index.html.en).Time of sunset for each observation day was obtained from the website of Ephemeris Computation Office (P. schwenkii were observed for a single fish each day for 5 days during 22nd May 2013 (11.6 age of moon) to 8th June 2013 (28.6 age of moon), which is proposed to be the spawning season for this species , and attached a chemical luminescent tag  to the caudal peduncle of the fish by using a cable-tie. We released the tagged fish into the school and monitored it for more than 10 min to confirm that the tagged fish rejoined the school and swam together with other individuals without physical damage and/or aggression towards the glowing tag  diving to the same depth as the tagged fish.The lat/log data recorded while tracing the movement of sweepers were plotted on the underwater topographic map. The marginal errors in the plotted data that arose due to mechanical mistakes were rectified as needed. Swimming direction and distance per 30 s recorded by GPS were used to estimate their general habit.3 and depths range from 2.5–6.6 m. It is continuously supplied with fresh seawater from the adjacent ocean mixed with circulated filtration seawater, thus the water temperature is almost comparable with that in the field. More than 200 fish species were kept in this tank, including almost 60 individuals of P. schwenkii. The tank was subdivided into six areas. The tank contains a large cave (station 1) which has two entrances connecting laterally and longitudinally with stations 2 and 6, respectively. A large artificial rock located at middle of tank. The circulated filtration seawater and fresh seawater supplied to station 4 and 6, respectively. Sweepers usually spent their daytime at cave (station 1) with locating near entrance facing station 2. The number of sweepers in each area and their behaviors were observed every min. The observations were carried out by three to five observers for a total of 3 days, throughout the night, from 18:30 of 30th May 2012 to 6:30 of 31st May 2012, and from 18:30 to 21:00 on 10th June 2012 and 13th June 2012. Light intensity was recorded by a data logger  just above the center of the tank. The tank was illuminated by a bright light, which was turned to low-intensity all-night light at 20:00. The light intensity gradually decreased until sunset, and went to almost zero when the main light was turned off.Observations were made in the “Tank of Tropical Sea” in Okinawa Cyuraumi Aquarium . The volPempheris schwenkii usually swam slowly in the open water deep inside the cave and formed one or two large schools. The preparation for nighttime migrations began at 13–35 min (mean: 21 min) after sunset when the entrance of cave was darker  after they left the entrance of the cave. When they reached the outer reef, the tagged fish mostly swam alone except for a few times when they met and swam together with other sweepers. Their migration patterns in outer reef differed day by day. In general, however, they spent almost all of their time in the area where the depth of sea bottom ranged from 5–30 m, and reached a maximum of 150 m offshore from the reef edge. In the offshore area (outer reef), the observer could not see anything except for a glowing tag, and perfect dark surrounding the fishes. They usually spent time near the surface water-column . The longest migration was observed on 8th June, when the sweeper swam straight to the east, and the shortest was observed at 31th May, when it spent most of the time in the deep water-column.After 1–18 min (mean: 7.4 min), the school suddenly started migrating towards the northeast (60–70°) to the outer reef in small group . The timP. schwenkii in the tank was categorized into five main patterns as follows  school in caves during daytime; (2) move to the entrance and wait for migration; (3) share a sign  when they spawn in the night; (4) quickly migrate from inner reef shelter to offshore outer reef; (5) spawn at outer reef (spawning season only); (6) disperse and feed on zooplankton in offshore open water-column for several kilometers; (7) return to their diurnal shelter before the day becomes bright.To the best of our knowledge, this was the first attempt to follow nocturnal fish under darkness using chemical luminescent tags. In this method, we could clearly observe the small-scale but dynamic nocturnal migration pattern of P. schwenkii is also supported by the gonadal data, because evening (15:00 to sunset)-collected specimens have the most developed ovaries in their gonads, and morning (sunrise to noon)-collected specimens most frequently exhibit postovulatory follicles, indicating they have recently spawned . This fact indicating that they are avoiding to spawn near their daytime shelter. In addition, the nocturnal migration observed in nature indicated that they immediately migrate to outer reef after they start migration. From these results, it can be inferred that they are likely to spawn after migrating to the outer reef. Spawning in the outer reef is well known in other fish groups, and reduces the risk of egg predation . The nig spawned . JohanneIn the aquarium, shaking behavior by all captive individuals was observed only on the day that spawning occurred. This behavior may be characteristic of spawning; however, more trials are warranted in this regard in the future. In spite of 60 individuals being kept in the aquarium and observations being carried out during their spawning season, the spawning behavior was observed only once in 3 days. This observation may be attributed to several possible reasons: they are too old for spawning (more than 8 years old), whereas the maximum age has been estimated to be 6 years old, and most of the individuals younger than 2 years old in nature ; the spaP. schomburgki. However, the present observation indicated that P. schwenkii usually swam in the shallow water column in the 20–30 m depth area, which is sometimes more than 100 m offshore from the reef edge. It may indicate that the darkness as perceived by human vision is still light enough for sweepers to recognize their position from the topographic landscape.Assuming they keep moving at the same speed and active nighttime is 10 h/day during the observed period, it was estimated that they can potentially migrate several kilometers  in one night. Even they if they immediately came back from the point we finished the observation to their home cave, it means they can potentially migrate at least 2 km in one night. It is well known that reef fishes are visually familiar with their surroundings, and often use landmarks during migrations . GladfelP. schwenkii wait at the entrance of cave for almost half an hour after the sunset, which indicates an attempt to reduce the predation risk from diurnal piscivorous fishes during this period. Therefore, they may start migration all at once after waiting for a while, which is deemed to have reduced the activities of nocturnal piscivorous fishes, and swim out to the offshore outer reef immediately. In our study, P. schwenkii swam near the surface or in the shallow water-column during the nighttime and were far away from the bottom or walls where there are structures that can shelter them from predators. In addition, they have no agility to escape from large piscivorous fishes, and neither do they have strong spines or scales to avoid predation. It is easy to imagine that they are preyed upon by large piscivorous fishes if they are found in the open water column at outer reef. In fact, members of the family Pempheridae are recorded from the stomachs of various large piscivorous fishes, such as Sphyrnidae, Serranidae, Carangidae, Lutjanidae, and Trichiuridae  of P. schwenkii was significantly higher than those of other reef fishes and that the species can grow up to more than 80% of their maximum length in the first year. In addition, P. schwenkii was not related to the lunar cycle, and occurred more frequently compared to other reef fishes. Their rapid growth and frequently reproduction may be attributed to their feeding behavior in that they can full up their stomach every night with rich zooplankton from outer reefs. The first author opened the fully filled stomachs of P. schwenkii collected early morning at the same cape, and found them filled with zooplanktons, such as Crustacea , Polychaeta, fish eggs, and larvae. Pempheris adusta was observed in the inner reef area during nighttime in the present observations and not at outer reefs. Previously, P. adusta was almost half of P. schwenkii, which might be a result of the difference in their feeding habits.On a coral reef many big fishes start to hunt at twilight. Most diurnal fishes fall asleep in this time zone, and are vulnerable to piscivores. hiuridae . It indihiuridae . The comhiuridae . Only a hiuridae . Koeda eChromis punctipinnis) which is a diurnal zooplanktivore in southern California waters (e.g., The observations in the nature and aquarium in the present study, combined with previous studies, clearly showed that sweepers inhabited in inner reef during daytime and migrated to an offshore outer reef to feed on rich zooplankton. This finding could be important in evaluating their role in the coral reef ecosystems. Firstly, they are considered to be an important prey item for the piscivorous fishes, which is usually located on the top of the coral reef ecosystem. Their growth rate is higher than many other groups , and thea waters . Howeverrs e.g., , and the10.7717/peerj.12412/supp-1Supplemental Information 1Mechanical mistakes were rectified.Click here for additional data file.


Metastasis occurs in the majority of pancreatic ductal adenocarcinoma (PDAC) patients at diagnosis or following resection. Patients with liver metastasis and those with lung metastasis have significantly different prognosis. Here, we sought to understand how cancer-associated fibroblasts (CAFs) play roles in the development of organ-specific metastasis.PDAC tumor cell lines established from the primary tumors with liver and lung metastasis potentials, respectively, in Kras/p53 mutation conditional knock-in (KPC) mice were co-cultured with matched CAFs or mouse mesenchymal stem cells. CAFs were isolated from metastases and subjected to DNA methylation and whole transcriptomic RNA sequencing analysis.The ability of mouse PDAC tumor cell lines in developing liver or lung-specific metastases was demonstrated in orthotopic models. Tumor cells associated with liver metastasis potential, but not those associated with lung metastasis potential, induced the methylation of metabolism genes including NQO1 and ALDH1a3 and subsequent downregulated mRNA expression of a broader group of metabolism genes in CAFs. DNA methylation and downregulation of metabolism genes in CAFs in liver metastasis, but not those in lung metastasis, appeared to be regulated by DNA methyltransferase. Tumor cells associated with liver metastasis potential, but not those associated with lung metastasis potential, induce inflammatory CAF (iCAF) signatures. CAFs from liver metastasis demonstrated a more homogenous iCAF phenotype, whereas CAFs from lung metastasis maintained the heterogeneity.PDAC with organ-specific metastatic potentials has different capacities in inducing methylation of metabolism genes in CAFs, modulating CAF phenotypes, and resulting in different levels of heterogeneity of CAFs in different metastatic niches.The online version contains supplementary material available at 10.1186/s13045-021-01203-1. To the editorALDH1a3 in the glucose metabolism pathway and NQO-1 in the oxidative phosphorylation (OXIPHOS) pathway  patients with different distant metastatic sites, resulting in distinct clinical outcomes . We sougNQO-1 and ALDH1a3 in moMSCs , the precursor of CAFs, by co-culturing with three different primary PDAC tumor cell lines derived from a KPC mouse without metastasis (PancPrimaryTumorCell), a KPC mouse with liver metastasis only (LiverMetTumorCell), and a KPC mouse with lung metastasis only (LungMetTumorCell). Remarkably, different tumor cell lines showed differential capacities in inducing DNA methylation on NQO-1 methylation was elevated in CAFs isolated from liver metastases compared to normal liver fibroblasts (Fig. NQO-1 and ALDH1a3 and downregulation of their mRNA expression or expression of their related metabolism genes in CAFs from liver metastasis could be reversed by treatment of DNA demethylating agent, decitabine, whereas methylation in CAFs from lung metastasis was not affected (Additional file We next sought to understand if tumor-induced gene methylation occurred in the metastatic niches where tumor cells metastasized to. We orthotopically implanted KPC cells with both liver and lung metastasis potentials Fig. a. The trsts Fig. b, but rests Fig. c. SupporNevertheless, it was unknown whether CAFs from different metastatic niches are functionally different. The functions of the subpopulations of CAFs were previously characterized and showed different transcriptomic signatures –10. In oAdditional file 1. Representative images of lung and liver metastases from KPC mice implanted with KPC tumor cells with lung and liver metatasis potentials and schema of how represntative metabolism genes were selected for study.


Zanthoxylum armatum is an important woody crop with multiple applications in pharmaceutics, cosmetics, and food industries. With continuous increases in the plantation area, integrated pest management is required for scale production when diseases caused by biotic factors such as pests and pathogens have become new problems, one of which is the infectious flower yellowing disease (FYD). Here, isolates of a new illarvirus (3) and a new nepovirus-associated subviral satellite RNA (12) were identified in Z. armatum, in addition to 38 new isolates of four previously reported RNA viruses. Sequence variation can be observed in viral/subviral quasispecies and among predominant isolates from the same or different samples and geographic origins. Intriguingly, RNA sequencing of different diseased trees invariably showed an extraordinary pattern of particularly high reads accumulation of the green Sichuan pepper-nepovirus (GSPNeV) and the satellite RNA in symptomatic tissues. In addition, we also examined small RNAs of the satellite RNA, which show similar patterns to those of coinfecting viruses. This study provides further evidence to support association of the FYD with viral/subviral infections and deepens our understanding of the diversity and molecular characteristics of the viruses and satellite, as well as their interactions with the host. Zanthoxylum, one of the most economically important genera in the family Rutaceae, comprises deciduous, spiny shrub species (Zanthoxylum armatum (Zhuye huajiao in Chinese), also known as green Sichuan (Szechwan) pepper, is a commercial species that generates green fruits that can be used as a seasoning for its special aroma and numbing flavor , that leads to a severe disorder of the floral organs, has emerged as the main restriction factor for the industrial development of Z. armatum  has been extensively applied for identification and detection of viruses present in various organisms, such as the research on all plant viruses in samples from single or multiple plant species under specific conditions . The gloZanthoxylum species that are transmissible among trees in agricultural environments and possibly pathogenic, and thus they are potential threats to the sustainability of the industry  with symptomatic and asymptomatic branches on a single plant. Six samples  from symptomatic and asymptomatic branches of three selected partial diseased Z. armatum trees, one from Yunnan Province and two from Chongqing Municipality, were collected for RNA sequencing (RNA-seq) and subsequent comparative analyses of viral/subviral species and accumulation. In addition, 14 samples, each from a completely diseased tree or tree without symptoms as a negative control were incorporated for viral/subviral diversity and variation analyses. In total, 20 samples were separately sequenced for etiology study of the FYD, in addition to a diseased tree (marked here as CQ1-D) with RNA-seq data from a previous study (A survey was first conducted in orchards to identify us study . Among tZanthoxylum genomes are publicly unavailable) used as references  annotate the contigs using a local BLASTx program with the viral sequence database (taxid:10239) downloaded from the National Center for Biotechnology Information (NCBI) used as a target.The collected tissues of each sample were used for total RNA extraction by the EASY spin Plus Complex Plant RNA Kit . The RNA was tested using a Nanodrop  and agarose-gel electrophoresis to ensure a high quality. After ribosome RNA depletion by the RiboZero Magnetic Kit , a library was built with the TruSeq RNA Sample Prep Kit . The treated RNA was then sequenced by the Mega Genomics Company  using an Illumina HiSeq X-ten platform with 150 bp layout in paired-end read length. Total small RNA (sRNA) was extracted with the EASYspin Plant microRNA Extract Kit , constructed as a library using the TruSeq Small RNA Sample Prep Kit (Illumina) and sequenced on an Illumina Hiseq2500 platform with a length of 50 bp per reading (Mega Genomics). A series of built-in programs in the CLC Genomics Workbench 11  were employed to process generated RNA-seq and sRNA-seq raw data in the following steps: (1) remove adaptors and low-quality reads, and the host-related reads by mapping to draft genomes of citrus within Rutaceae family  de To exclude the possibility of chimeric viral/subviral sequences that result from assembly program algorithm error and false virus-positive results caused by contamination during HTS, contigs of the new ilarvirus from CQ7-DS and the satellite from CQ1-D were verified by RT-PCR using the leaf RNA extract, viral/subviral contig-specific primers , and a O1, and conserved protein domains were searched with the Conserved Domain Database (CDD) web tool2. Sequence comparison was conducted in the CLC Genomics Workbench 11 to identify conserved sites among viral/subviral sequences.A heatmap of the read numbers of viral/subviral RNAs in different samples (clustering by the squared Euclidean distance) was drawn by Heat Map with Dendrogram extension in Origin 2017 software , and a flower plot of common (center) or unique  viral/subviral RNAs across symptomatic or symptomatic/healthy samples was drawn by R (ver. 4.0.2) using draw.ellipse and draw.circle functions according to previous research . Open reViral/subviral nucleotide (nt) and amino acid (aa) sequences from this study and the GenBank databank were aligned using MAFFT 7 ; poorly The small RNA data previously used were reanalyzed to extract sRNA characteristics of the new satellite RNA that is likely associated with the nepovirus . Reads wNepovirus genus-associated satellite RNA and viruses in the genus Ilarvirus (family Bromoviridae), based on the e-values  from BLASTx analysis. After verification by RT-PCR, cloning, and sequencing of complete nucleotide sequences of the satellite and most of the ilarvirus genome and based on preliminarily taxonomic analyses, we tentatively named the satellite and ilarvirus “green Sichuan pepper-nepovirus large satellite RNA” (satGSPNeV) and “-ilarvirus” (GSPIlV), respectively. With the use of GSPNeV, satGSPNeV, GSPIdV, and GSPEV from the CQ1-D sample and GSPIlV from the CQ7-DS sample as reference genomes for read mapping, viral/subviral consensus sequences between the references and mapped reads from other samples were obtained. Since viral/subviral nucleotide sequences derived from independent assembly of all sample data were highly identical to the consensus sequences (>99%), these consensus sequences were considered predominant and accurate and were directly used for other analyses. If high sequence heterogeneity is present in a viral/subviral quasispecies that can affect sequence assembly, contigs from assembly and consensus sequences from read mapping with different parameters  will be obviously distinct from one another, and our tests showed that the viral/subviral sequences resulting from different conditions were highly consistent.After data processing, a total of 49,756,462 (6.95 Gb)–84,446,876 (11.8 Gb) clean reads were obtained for individual sequencing of 20 samples except that of CQ1-D, which was previously published . The reaOverall, reads from GSPNeV and satGSPNeV were more abundant in diseased samples, in which they accounted for 12–36% of the total reads relative to the reads from all other viruses , but in those samples for which symptoms were not obvious, the read proportions for GSPNeV and satGSPNeV were < 0.003% . A more Ilarvirus, especially the subgroup III (G3) members  was partially sequenced, and its nearly complete genomes are tripartite, exhibiting canonical genomic organizations found in the genus  members , that is members . Extensi members . RNA1 (∼ members . In addi members . We prope = 1.85E–10) and chicory yellow mottle virus large satellite RNA (e = 8.71E–23). The phylogenetic analysis with the complete nucleotide sequences also placed satGSPNeV with these two satellite RNAs  was obtained, which comprises 2,247 nts excluding the poly(A) tail. Its genomic 5′ and 3′ termini are to some extent conserved compared with GSPNeV RNAs . Two ORFite RNAs , on leftite RNAs . HoweverBased on the viral/subviral consensus sequences (or preponderate sequences) of quasispecies obtained from the read mapping of available samples, phylogenetic dendrograms of the viral/subviral RNAs from different isolates were constructed to gain insight into their co-evolution  and co-transmission  relationships by weighting both phylogenetic and geographic discrepancies between the trees . The popr > 0.77 .Viral/subviral isolates from CQ1-D and CQ7-DS samples were used as reference genomes, based on which nucleotide sequence variation sites from comparisons with other available conceptional isolates (the consensus sequences) were counted and added together  except for several hotspots . Among satellite .Z. armatum production in China after an endemic outbreak turned invasive and prevalent in the southwestern regions, including Chongqing Municipality, Yunnan, and Sichuan provinces, the main producing areas. A previous study revealed four different RNA viruses in one diseased tree (named CQ1-D in this study), and that GSPNeV was mainly associated with the FYD through field investigations (Ilarvirus and the subviral category, respectively (Z. armatum were still unclear due to the limited number of infected samples investigated. The relationships between these non-FYD-related viruses and the hosts, i.e., competition or mutualism, remain unknown.Over the last ten years, the FYD has become a restrictive factor in igations . Here, tectively . SatGSPNectively , likely Z. armatum.In agroecosystems, plant viruses sometimes are destructive to crop industries because of the presence of sensitive cultivars . In factGenomic variation site analyses of the viruses and satellite allowed the assessment of their adaptability to different environments. Long- and short-term variation sites predicted from the comparisons of different consensus sequences and quasispecies, respectively, were largely discrepant in spite of a similar tendency in quantitative changes, suggesting the presence of circumstance-specific variations in the quasispecies. More strong evidence was obtained directly from observations of variable site patterns of the quasispecies from different locations. Nevertheless, we have to note that there are selective processes during viral evolution, so short-term variation sites are time-sensitive, changeable at different stages, and may not be ultimately preserved . HoweverCutting off of the transmission routes of viruses, if they do exist, is a simple but practical method for management and control before viruses lead to greater losses . Apart fZ. armatum and helpful for understanding viral/subviral evolution. Then, our future works will have a shift of emphasis toward assessing biological properties of GSPNeV and satGSPNeV by construction of their infectious cDNA clones in planta. Meanwhile, parallel work is urgently needed, regarding elucidating the viral/subviral transmissions in fields, to restrain the dispersals.In summary, based on HTS-dependent viromic analyses, we discovered a new satellite RNA associated with GSPNeV and the FYD, as well as a new ilarvirus, and we studied their regional viral/subviral diversification, as well as that of other RNA viruses. This information can be useful for sustainable development of MH323432–MH323437 and MW962309–MW962391, and in the NCBI SRA repository, BioProject PRJNA732832, BioSample SAMN19341721–SAMN19341741, SRA accession numbers SRR14663372–SRR14663392.The data presented in the study are deposited in the NCBI GenBank repository, accession numbers MC conceived and designed the experiments. SZ, RL, XW, ZX, BZ, ZL, JZ, XD, ZT, SL, and YZ collected the samples and conducted the experiments. MC and SZ analyzed the data. MC, SZ, SL, and YZ discussed the results, drafted, and revised the manuscript. All authors read and approved the final draft of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CRISPR-LwCas13a system could detect BVDV virus and BVDV RNA with high specificity and simplicity. The detection limit of the LwCas13a system was 103 pM, and there were no cross-reactions with HEK293T and MDBK. In summary, a sensitive, specific, and simple nucleic acid detection method based on CRISPR-Cas13a was developed for BVDV. This method provides a new detection strategy for early diagnosis of BVDV.Bovine Viral Diarrhea Virus (BVDV) is the main pathogen of bovine viral diarrhea disease (BVD), which leads to enormous economic losses in the cattle industry. A sensitive and specific detection for BVDV is advantageous to the control of BVDV. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have been used for detecting virus RNA. In this study, the expression and purification of LwCas13a protein was optimized and the RNase activity of LwCas13a  Pestivirus genus and Flaviviridae family  is a high-prevalence viral of cattle, affecting herds worldwide and leading to significant economic losses in the cattle industry . BVDV ise family . The dise family –5. In ade family . Thus, ce family . At prese family . Traditie family , so it iin vitro experiments found that, after complementary pairing of crRNA and target single-stranded RNA, Cas13a was combined to form a complex and underwent a conformation change, activating non-specific RNase activity that could target cleavable accessible single-stranded RNA (ssRNA) (Leptotrichia wadei Cas13a (LwCas13a) that activated non-specific RNase activity could detect Zika and Dengue viruses in samples  based nucleic acid detection platforms  with hig (ssRNA) –20. Amon samples . Similarin vitro was measured. The sensitivity and specificity of the LwCas13a-based detection system was evaluated. A new nucleic acid detection method for the BVDV virus has been developed, which provides a new strategy for field detection of BVDV and an efficient and practical platform for RNA virus detection.In this study, our aim is to develop a CRISPR-Cas13a-based diagnostic technology system for BVDV detection. A pair of specific crRNAs was designed based on the reported BVDV gene sequence in the 5'UTR conserved region, and Reporter (quencher fluorescent RNA) was synthesized. The expression and purification of LwCas13a protein were optimized, and the RNase activity of LwCas13a M31182) was preserved in our laboratory.The Twinstrep-SUMO-huLCas13a plasmid was generously donated by Zhang Feng Laboratory . The MDBK cell line and HEK-293T cell line were purchased from the Chinese Academy of Sciences. The BVDV . Design of LwCas13a Protein Full-length Primers was carried out with Premier 5 . The primers were synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd.  preparation, the design of crRNA was carried out according to NADL strain select BVDV sequence that contains the highly conserved sites of the 5'UTR region, and these were then were synthesized by TaKaRa  Co., Ltd. . The flu+ . And then, the LwCas13a recombinant Plasmid was transformed into Rosetta™ (DE3) pLysS Singles Competent Cells . Sixteen milliliters overnight starter culture was added into Terrific Broth 4 L growth media  (TB). Subsequently, LwCas13a was inoculated at 37°C and 200 rpm until an OD600 of 0.6. Meanwhile, the culture was supplemented with 500 uM  IPTG  and induced for protein expression at 18°C for 20 h.LwCas13a protein purification was performed as previously described with some modifications . The fulThe culture was centrifuged at 8,000 rpm for 15 min at 4°C and the cell pellet was then collected. The cell pellet was then crushed and suspended in lysis buffer , and then supplemented with Lysozyme (Sigma), benzonasefor® Nuclease, and protease inhibitors (Complete Ulter EDTA-free tablets) followed by sonication (Ningbo Xinzhi Scienz-IID) under the following conditions: power 60%, 2 s on and 3 s off, the total sonication time is at least 30 min until the lysate cleared. It was then centrifuged for 30 min at 4°C at 12,000 g and the supernatant was harvested.2PO4, 500 mM NaCl, 500 mM imidazole pH 7.4) according to the operation method as specified in the manual . This was confirmed by SDS-PAGE and Coomassie blue staining. After, the elution buffer was concentrated on protein storage buffer  by centrifugation filter unit (Millipore ultrafiltration centrifuge tube). The LwCas13a protein was stored at −80°C.The supernatant was filtered through 0.22 uM and applied to 5 mL HisTrapTM crud FF. Then, the bound protein was eluted with buffer  was added into the cell culture medium and allowed to culture until more than 80% of the cells' pathological changes. Finally, virus solution was collected using Trizol (Thermo Fisher Scientific) and was stored at −80°C until use.MDBK cells and HEK293T cells were cultured in high-glucose Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum (FBS)  (GM) at 37°C with 5% COThe viral RNA of BVDV was extracted using QIAamp Viral RNA Mini Kit  according to the manufacturer's instruction. Subsequently, total RNA was reverse transcribed into cDNA using the Prime Script RT Master Mix  according to the manufacturer's instruction. MDBK cells and HEK293T cells were used to extract the RNA. All RNA and cDNA were stored at −20°C until use.We first verify the activity of the LwCas13a protein. The biochemical characterization of LwCas13a revealed crRNA mediated RNA cleavage promoted by two HEPN nuclease domains. Upon recognition of its RNA target, crRNA and Cas13a can bind to complement and trigger LwCas13a, which engages in “collateral” cleavage of nearby non-targeted RNAs. As a result, the fluorescence (FAM)-and quencher (BHQ-1) -labeled RNA probe in 2, pH 7.3). The total reaction volume was 40 uL. The mixture was incubated in a 96-well plate on Synergy HTX Multi-Mode Reader  at 37°C for 1 h with fluorescence kinetics measurements  taken every 5 min.The detection system included: 45 nM purified LwCas13a, 22.5 nM crRNA, 125 nM RNA Reporter , 2 uL murine RNase inhibitor (New England Biolabs), 100 ng of background RNA (purified from HEK293T), varying amounts of BVDV RNA, and LwCas13a detection buffer . The directly extracted RNA cannot be detected with sensitivity due to partial degradation or loss during the process and because it contains multiple RNA fragments. Therefore, we used real-time fluorescence quantitative PCR (RT-qPCR) to make a set of standard curves for the standard RNA, and used the standard curve to quantify the virus sample RNA. Subsequently, the expressed RNA standard was diluted at a 10-fold ratio, and five diluted components were selected as the standard for RT-qPCR. All the samples of each condition were three biological replicates. The RNA concentration of the obtained BVDV virus sample was divided into eight groups by a 10-fold dilution method for the determination of the sensitivity of the CRISPR-Cas13a system.All data in this study was analyzed using the one-way analysis of variance (ANOVA) and GraphPad Prism 8 software  for drawing analysis. The pictures in the article were processed by Photoshop CS6 .+ vector. As shown in +-LwCas13a could be digested into two fragments. The resulting fragment had an expected size and was verified by DNA sequencing. Then, the recombinant plasmid was transformed into Rosetta™ (DE3) for analysis of the LwCas13a protein expression. As shown in The full-length sequence of LwCas13a  was amplified by PCR and cloned into the pET-28aWe next tested the activity of the expressed LwCas13a protein, and the complete LwCas13a reaction system were assessed. As shown in We tested the specificity of the LwCas13a system for BVDV detection. We used HEK293T, MDBK, and BVDV virus as controls to detect specificity. The rapid reaction against BVDV virus and BVDV RNA occurred, and there was no cross-reaction with controls . The res8 pM down to 103 pM template could be detected. These data showed that the detection limit of the LwCas13a system was 103 pM.We tested the detection sensitivity of the LwCas13a detection system. The sensitivity of the LwCas13a detection system was tested with 10-fold serial diluted template BVDV (NADL). As shown in Bovine Viral Diarrhea Virus (BVDV) is an economically significant viral disease in the global cattle industry. Since the first identification in China in 1980 , BVDV haSequence analysis revealed that LwCas13a lacks an identifiable DNase catalytic site, and two HEPN domains containing highly conserved R-X-H motif were identified . FurtherZhang Feng's research is based on LwCas13a molecular detection platform (SHERLOCK), which can distinguish between Zika virus and Dengue virus at low concentrations . AlthougIncreasing evidence has indicated that combining Csm6 with LwCas13a detection can improve the stability of the test and reduce the possibility of false positive readings . FurtherWe have established a method for detecting the NADL strain based on CRISPR-Cas13a. Although the method needs to be constantly improved and strengthened for visualization and field testing, it opens up a new strategy for rapid, stable, and sensitive detection of RNA viruses such as BVDV.The original contributions generated for the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.All experimental procedures in this study were carried out in accordance with the requirements of the Statute on the Administration of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee at Shihezi University (SU-ACUC-08032).RY, YX, LW, DW, WN, and SH conceived and designed the experiment and analyzed and interpreted the data. RY, YX, LR, CL, WN, and SH wrote the manuscript. RY, YX, CL, WN, SH, XL, RH, TG, YL, LL, and XW took part in the expression and purification of LwCas13a protein. RY, YX, and LW participated in RNA extraction and the construction of CRISPR-Cas13a detection system and processed the data. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lentiviral (LV) vectors have emerged as powerful tools for transgene delivery ex vivo but in vivo gene therapy applications involving LV vectors have faced a number of challenges, including the low efficiency of transgene delivery, a lack of tissue specificity, immunogenicity to both the product encoded by the transgene and the vector, and the inactivation of the vector by the human complement cascade. To mitigate these issues, several engineering approaches, involving the covalent modification of vector particles or the incorporation of specific protein domains into the vector’s envelope, have been tested. Short synthetic oligonucleotides, including aptamers bound to the surface of LV vectors, may provide a novel means with which to retarget LV vectors to specific cells and to shield these vectors from neutralization by sera. The purpose of this study was to develop strategies to tether nucleic acid sequences, including short RNA sequences, to LV vector particles in a specific and tight fashion. To bind short RNA sequences to LV vector particles, a bacteriophage lambda N protein-derived RNA binding domain (λN), fused to the measles virus hemagglutinin protein, was used. The λN protein bound RNA sequences bearing a boxB RNA hairpin. To test this approach, we used an RNA aptamer specific to the human epidermal growth factor receptor (EGFR), which was bound to LV vector particles via an RNA scaffold containing a boxB RNA motif. The results obtained confirmed that the EGFR-specific RNA aptamer bound to cells expressing EGFR and that the boxB containing the RNA scaffold was bound specifically to the λN RNA binding domain attached to the vector. These results show that LV vectors can be equipped with nucleic acid sequences to develop improved LV vectors for in vivo applications. Over the past two and a half decades, lentiviral (LV) vectors have emerged as powerful tools for transgene delivery . LV vectLV vectors are also applied directly in vivo for therapeutic purposes. A non-primate LV vector system based on equine infectious anemia virus (EIAV) has been investigated clinically to treat ocular disorders . In anotThe design of LV vectors to allow the targeted transduction of specific cell types in vivo has been challenging and the efficacy of such vectors can be affected by neutralizing antibodies. Virus engineering approaches have been critical in helping to address some of these issues ,11,12. FEngineering strategies aimed at the enhancement of the purification of LV vectors have also been investigated. For example, Yu et al.  used a lSurface modification approaches have also been used to dampen innate immune responses to LV vectors . LV vectOther approaches to the modification of the surface of LV vectors have included direct covalent modification. Shielding of VSV-G-pseudotyped LV vectors by poly(ethylene) glycol (PEG) conjugation (PEGylation) was shown by Croyle et al.  to increThe goal of this study was to develop novel approaches to tether short RNA sequences to LV vector particles in a specific fashion, using RNA binding proteins. The bacteriophage λN protein-boxB system  providedTo attach RNA sequences to LV vector particles, a 22 amino acid RNA binding domain (λN) derived from the bacteriophage lambda antitermination protein N ,32 was uIn our approach, the λN domain bound to a boxB sequence present on a scaffold RNA . The scaTo display λN protein domains on the HEK 293T cells, a recombinant plasmid encoding two copies of an affinity-enhanced λN protein domain, referred to as λN22  , was useCompared to the HEK 293T cells expressing the H-λN2 protein, only a minor increase in the PE signal was observed in the cells transfected with control plasmids expressing the MV H protein fused either to IL-13 or EGF B. FurtheTo determine the scaffold binding to the LV vector particles, a mutated version of the MV H protein ,36 boundThe EGFP-encoding LV vector particles pseudotyped with the H-λN2 (or H-EGF) and VSV-G glycoproteins were used to monitor the transduction of human skin epidermoid carcinoma epithelial A431 cells A. The peTo test the ability of the RNA scaffolds described above to bind to other RNA sequences, a J18 RNA aptamer-based sequence was used. The J18 aptamer was previously shown to bind to human EGFR ,39,40. TAn RNA aptamer (SA19) directed against streptavidin  was usedThe J18 Rvs aptamer, a modified version of the J18 aptamer, was used to assess the scaffold binding. It was generated by in vitro transcription, using the DNA template shown in The J18 Rvs aptamer also bound to rat F98 cells expressing human EGFR (referred to as F98/EGFR cells) , but notThe capacity of the various scaffold RNAs to bind to λN protein sequences was tested next. To do this, aptamer/scaffold complexes were mixed with a synthetic λN peptide bound to FITC (referred to as λN-FITC peptide) A. The A4The binding specificity of the aptamer/scaffold/λN-FITC peptide complex was assessed in more detail by comparing the ability of the J18 Rvs aptamer/Ab2bA RNA scaffold/λN-FITC complex and the SA19 aptamer/Ab2bA RNA scaffold/λN-FITC complex to bind to A431 cells and to the MDA-MB-435 cells that lacked EGFR . The resIt was interesting to note that the RNA scaffold binding to the H-λN2 protein displayed on the LV vector particles was highest with the Ab and bA scaffolds B, while Different protein ligands, such as EGF , IL-13 ,44, singMethods aimed at the attachment of short nucleic acid sequences to LV vector particles in a specific and tight fashion have not been reported thus far. The work described in this study describes a novel approach to the tethering of RNA aptamers to LV vector particles based on the bacteriophage λN/boxB system . The bacThe bacteriophage MS2 coat protein system is the most widely used RNA tethering system . The popOur results show that the λN/boxB system provides a promising approach to the attachment of short RNA sequences to LV vector particles. In addition, it has the potential to bind short DNA sequences, including DNA aptamers, via a boxB-containing complementary RNA. Our results also show that the binding of the various boxB-containing RNA scaffolds and aptamer sequences to λN protein sequences was specific. In addition, the H-λN2 protein displayed on the LV vector particles, along with the VSV-G, allowed the transduction of A431 cells. Replacing the VSV-G with smaller fusion domains, including the VSV-GS domain  may ultiParamyxoviridae family, including those derived from the Tupaia paramyxovirus (TPMV) have also been used to pseudotype LV vectors [The limitation of the RNA-capturing approach described in this study is that the high prevalence of antibodies against measles virus proteins may hamper the efficacy of LV vectors containing engineered measles virus H glycoproteins, including H-λN2, in a clinical setting, due to vaccination or natural infection . Glycopr vectors . The abs vectors . The use vectors . The imp vectors . CpG seq vectors .In future studies we plan to determine the usefulness of RNA and DNA aptamers tethered to LV vector particles via the λN protein-boxB system for targeted transduction in vitro and in vivo ,54. We aA synthetic DNA sequence  encoding the λN2 protein (GNAKTRRRERRAEKQAQWKAANGAGAGAGAGAGAGNAKTRRRERRAEKQAQWKAAN) ,35 was sThe pNL (CMV)EGFP/CMV/WPREΔU3 LV vector plasmid was described previously . It is aThe A431 cells , MDA-MB-435 cells (ATCC HTB-129), HEK 293T cells (ATCC CRL-3216), F98 cells and F98/EGFR cells  were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) containing high glucose (4.5 g/L), 2 mM L-glutamine, 10% heat inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin. The cell culture reagents were purchased from Gibco . 6 293T cells were seeded into each 15 cm plate. The vector production was carried out via PEI-mediated transfection [g for 45 min at 4 °C. The pellets were resuspended in 1/10th of the original volume using a 100 mM HEPES buffer (pH 7.5) plus 0.2 U/µL of RNase inhibitor . By contrast, the vectors were concentrated by ultracentrifugation, as described by Kutner, et al. [The LV vector production was carried out using 15-cm plates . One day before transfection, 6 × 10sfection  of 7.65 sfection , 5.1 µg sfection , 1.95 µgsfection  and 0.6 sfection  per 15 c, et al. . TM RNase inhibitor (Lucigen) (40 U/tube of in TE buffer) was added. The samples were incubated overnight at 37 °C with constant shaking. The extracted RNAs were recovered by filtration, using a 0.45 µm filter , followed by ethanol precipitation. The RNA pellets were resuspended in the TE buffer. By contrast, the RNAs were purified and concentrated using an RNA Clean & ConcentratorTM −5 kit . The recombinant plasmids encoding the RNA scaffold and the aptamer sequences were linearized using the restriction enzymes referred to in For cell binding, the J18 RNA aptamer (5′ GGCGCUCCGACCUUAGUCUCUGCAAGAUAAACCGUGCUAUUGACCACCCUCAACACACUUAUUUAAUGUAUUGAACGGACCUACGAACCGUGUAGCACAGCAGAGAAUUAAAUGCCCGCCAUGACCAGAU 3′) was bound to a complementary single-stranded DNA oligonucleotide bearing a biotin group at the 5′ end , using RNA/DNA hybridization. The oligonucleotide used is referred to as oligo Bio (5′ Bio-TGGTCATGGCGGGCATTTAATT 3′). The J18 Rvs aptamer, a modified version of the J18 aptamer, was used to assess the scaffold binding. It consisted of the original J18 aptamer sequence but contained a different sequence (5′ GUGGUCAUGGCGGGCAUUUAAUU 3′) at its 3′ end. To investigate the binding of the J18 Rvs RNA aptamer to the cells, a complementary single-stranded DNA oligonucleotide, referred to as Bio oligo (5′ Bio-AATTAAATGCCCGCCATGACCA 3′) was used. One-to-one molar ratios of the oligo Bio plus the J18 aptamer, or the Bio oligo plus the J18 Rvs aptamer were used. The controls included an RNA aptamer (SA19) directed against streptavidin   , using a molar ratio of 2:1. To analyze aptamer binding, A431, MDA-MB-435 cells, F98/EGFR cells or F98 cells were trypsinized using 0.25% Trypsin-EDTA (Thermo Scientific) and washed three times with a DPBS/5 mM MgCl2O/0.1% BSA were added to compete for aptamer binding to the EGFR. An aptamer that does not bind to EGFR (SA19) was used as an additional control for specificity.To assess the specificity of the J18 aptamer binding, rEGFR  or rEGF  in H5 HEK 293T cells per well were seeded in six well-plates. The next day, the medium was replaced and 1 µg of the pCG-HcΔ18-AA-λN2, pCG-HcΔ18-AA-IL-13 or pCG-HcΔ18-AA-EGF plasmid DNAs in 150 mM were mixed at a 1:1 (v/v) ratio with 12.6% PEI in 150 mM NaCl, and the DNA/PEI mixture was added to the wells [++, and Mg++ free PBS containing 0.5 mM EDTA and 50 mM NaCl. The cells were washed three times using PBS and 2 × 105 cells were incubated in the dark for 30 min at room temperature, using a 50 nM solution containing the RNA scaffold base-paired to the Bio oligo, which was bound to SA-PE in a 50 mM HEPES buffer pH 7.5 . After washing using the 50 mM HEPES buffer, the cells were analyzed using flow cytometry.To test the ability of the various RNA scaffolds to bind to MV H-λN2 proteins displayed on the surface of cells, the HEK 293T cells were transfected using the pCG-HcΔ18-AA-λN2 plasmid. The plasmids pCG-HcΔ18-AA-IL-13 and pCG-HcΔ18-AA-EGF were used as controls. The day before transfection, 2 × 10he wells . Forty-e2, was mixed with 100 µL of a 30 µM solution of the λN peptide conjugated to FITC (FITC-GNAKTRRRERRAEKQAQWKAAN) (Genscript) in DPBS/5 mM MgCl2, and incubated for 20 min at 25 °C. For the cell binding, 60 µL of the J18 Rvs aptamer/RNA scaffold/λN-FITC complex were added to 2 × 105 trypsinized A431 or MDA-MB-435 cells and the cells were incubated for 30 min at room temperature in the dark. To determine the specificity of the aptamer binding, 6 μL of a 0.1 µM rEGFR  solution in H2O/0.1% BSA were added to compete for aptamer binding to the EGFR on the A431 cells. Treatment with 0.02 U/µL of RiboShredderTM RNase Blend (Epicentre) on ice was used to confirm that λN peptide-FITC binding was mediated by the RNA. After incubation for 30 min at room temperature on ice, the cells were washed three times and analyzed using flow cytometry. As an additional control, the SA19 aptamer that does not bind to EGFR was used.The RNA aptamers and scaffolds were mixed and base pairing was initiated via the conditions used for the binding the J18 Rvs aptamer to the Bio oligo. To do this, 100 µL of a 0.15 µM solution containing the J18 Rvs aptamer, plus the RNA scaffold in DPBS/5 mM MgCl® M-280, Thermo Scientific). To do this, the beads (20 µL/sample) were washed three times with DPBS/5 mM MgCl2 and incubated with a ~100 nM solution of a particular RNA scaffold base-paired to the Bio oligo in the presence of 1.3 U/µL of RiboGuardTM (Lucigen). After shaking for 20 min at 1300 rpm at room temperature, a 100 µL aliquot of the magnetic beads containing RNA scaffolds was mixed with a 100 µL aliquot of a LV vector sample displaying either H-λN2 or H-EGF. The titers of the LV vectors displaying the H-λN2 or H-EGF proteins plus VSV-G were typically around 2 x 106 transducing units per ml. The samples were incubated for one hour at 4 °C, with gentle vortexing every 10 min. The samples were then washed three times and the magnetic beads with bound vector particles were diluted in DMEM containing 8 µg/mL polybrene and added to the A431 cells in six wells-plates, seeded the day before, at a density of ~1 × 105 cells/well. The percentage of transduced cells based on EGFP expression was determined using flow cytometry three days later [The lentiviral vector particles bearing H-λN2 proteins were captured using streptavidin-coated magnetic beads (Dynabeadsys later .


Quality control checks are the first step in RNA-Sequencing analysis, which enable the identification of common issues that occur in the sequenced reads. Checks for sequence quality, contamination, and complexity are commonplace, and allow users to implement steps downstream which can account for these issues. Strand-specificity of reads is frequently overlooked and is often unavailable even in published data, yet when unknown or incorrectly specified can have detrimental effects on the reproducibility and accuracy of downstream analyses.To address these issues, we developed how_are_we_stranded_here, a Python library that helps to quickly infer strandedness of paired-end RNA-Sequencing data. Testing on both simulated and real RNA-Sequencing reads showed that it correctly measures strandedness, and measures outside the normal range may indicate sample contamination.https://github.com/betsig/how_are_we_stranded_here.how_are_we_stranded_here is fast and user friendly, making it easy to implement in quality control pipelines prior to analysing RNA-Sequencing data. how_are_we_stranded_here is freely available at The online version contains supplementary material available at 10.1186/s12859-022-04572-7. Common sequencing design of RNA-Seq libraries are either paired-end, where fragments are sequenced from both the 3RNA-Sequencing (RNA-Seq) is the mentclass2pt{minimFurther, library preparation protocols for RNA-Seq can be stranded or unstranded. In unstranded libraries, no information is preserved about the original transcript orientation. In contrast, stranded protocols retain strand information by attaching adapters, or through chemical modification of RNA or the paired cDNA during library preparation . StrandeDownstream RNA-Seq processing pipelines often incorporate information about library design in the workflow, e.g., via a strand-specificity (or strandedness) parameter in RNA assembly and read counting tools. Incorrect use of this parameter can significantly impact the output of RNA-Seq analyses. For example, defining a stranded library as unstranded can result in over 10% false positives and over 6% false negatives in downstream differential expression results .RNA-Seq sample strandedness and direction of strandedness is not available as metadata for RNA-sequencing samples in repositories such as The European Nucleotide Archive (ENA) or Sequence Read Archive (SRA), and in the cases where there is a corresponding paper, is often not reported in the methods. From a randomised investigation of 50 ENA “PAIRED END” studies with an associated publication, we found only 56% have strandedness either explicitly stated or mentioned in the methods section for library preparation  and IRFhow_are_we_stranded_here is written in Python3 and runs a series of commands to determine read orientation. First, a kallisto  index ofWe first tested how_are_we_stranded_here on simulated samples across three species—human (Homo sapiens), yeast (Saccharomyces cerevisiae), and thale cress . Using lower numbers of reads resulted in greater variation in percent stranded reads for samples with non-strand-specific reads  or less than 0.6 (unstranded), when removing data points where less than 10% of sampled reads aligned to the transcriptome Fig. . ReporteWhile the majority of samples matched their reported strand-specificity, there were seven which did not  should be detailed in the methods of published RNA-Seq data, as this is vital to being able to reproduce results, we found that over a third of publications do not provide this detail.how_are_we_stranded_here allows users to easily find the correct strandedness parameter for RNA-Seq datasets which is crucial for reproducibility of published results.All read runs matching the rules library_strategy=“RNA-Seq”, library_source=“TRANSCRIPTOMIC”, and library_layout=“PAIRED” were retrieved from ENA . The runAll read runs matching the following rules were retrieved from ENA for each human , yeast  and thale cress : instrument_platform = “ILLUMINA”, library_strategy=“RNA-Seq”, library_layout=“PAIRED”, library_source=“TRANSCRIPTOMIC”, library_selection=“cDNA”. These runs were then filtered for those sequenced on an Illumina HiSeq 2000, 2500, 3000, or 4000, between 10 and 30 samples per study, and randomly reordered. Studies were searched for any publications that were associated with the data, and only retained if strandedness was stated or able to be inferred by the library preparation methods (see above). The first 20 studies matching these requirements were used for analyses for each species.Reads were simulated using polyester . KallistFor each study the first three samples were taken to profile for strandedness. A kallisto  index waAll steps were performed on samples of 200,000 reads from each fastq file, to match the number of reads used for how_are_we_stranded_here. FastQC 0.11.5 (Babraham Bioinformatics) was used to assess the quality of reads, and Trimgalore! 0.6.0 (Babraham Bioinformatics) in paired end mode to quality trim reads and remove adapter sequences. We used FastQ Screen 0.14.0 (Babraham Bioinformatics) to screen for contaminants in trimmed fastq files, with the default databases downloaded by “fastq_screen–get_genomes”. Reads pairs which both mapped only to the correct genome were extracted for each sample using the—tag flag in FastQ Screen, and a custom R script (see Code availability). We then ran how_are_we_stranded_here on the trimmed, and the exclusively correctly-mapping fastq files. For genome alignment with STAR 2.7.0e , the firAdditional file 1. Figure S1: Strandedness proportions in simulated data. Figure S2: Reads mapping to intergenic regions in s. cerevisiae samples. Figure S3: Strandedness proportions are not altered by trimming or filtering readsAdditional file 2. Table S1: Library preparation and strand specificity reporting in paired-end RNA-Seq studies. Table S2: Differences in variation of reported how_are_we_stranded_here strand-specificity values using increasing numbers of reads. Table S3: Run times for how_are_we_stranded_here using the default 200,000 reads on a 2020 M1 Macbook Pro. Table S4: how_are_we_stranded_here results on RNA-Seq data. Table S5: List of RNA-Seq studies with strand specificity searched in methods. Table S6: Proportion of reads aligning to different genomes with fastqscreen