However, the contribution of these two RNA modifications to the development, prognosis, regulation mechanisms, and drug sensitivity of gastric cancer (GC) is unclear.Most human genes have diverse transcript isoforms, which mainly arise from alternative cleavage and polyadenylation (APA) at 3′ ends. N7‐methylguanosine  to quantify each GC patient's RNA modification index. Finally, we examined the correlation between RMscore and clinical features such as survival outcomes, molecular subtypes identified by the Asian Cancer Research Group (ACRG), posttranscriptional regulation, and chemotherapeutic sensitivity in GC.The samples were categorized into two groups on the basis of their RMscore: high and low. The group with a low RMscore had a bad prognosis. Moreover, the low RMscore was associated with KRAS, Hedgehog, EMT, and TGF‐β signaling, whereas a high RMscore was related to abnormal cell cycle signaling pathway activation. The findings also revealed that the RMscore contributes to the regulation of the miRNA‐mRNA network. Drug sensitivity analysis revealed that RMscore is associated with the response to some anticancer drugs.7G‐RNA modifications may reveal novel insights into the epigenetics of GC and aid in the development of more effective treatment strategies.The RMscore model has the potential to be a useful tool for prognosis prediction in patients with GC. A comprehensive investigation of APA‐RNA and m We constructed an RNA modification model (RMscore) to quantify the RNA modification index of each GC patient. The RMscore model is a potentially useful tool to predict the prognosis of GC. Comprehensive analysis of APA‐RNA and m7G‐RNA modifications may provide novel insights into the epigenetics of GC and improve treatment strategies. Furthermore, these two RNA modification mechanisms regulate mRNA splicing, cellular location, transcription, and translation in the mRNA life cycle.The 5′ cap and 3′ poly(A) tail are two major common posttranscriptional modifications of mRNA, which are responsible for most of the various isoforms by generating alternative modification sites at the 5′ and 3′ ends.7G is co‐transcribed at the 5′ cap during transcriptional initiation to ensure transcriptional stability and efficiency.7G in mRNA and identified METTL1/WDR4 as an m7G writer complex, which affects the translation of mRNA.7G‐RNA modifications.Human genes (at least 70%) express multiple polyadenylation sites, and APA events are reported to be important triggers for many cancers.7G‐RNA modification could provide novel insight into the mechanisms underlying the development and tumorigenesis of GC. The APA‐RNA and m7G‐RNA modifications were used to develop a prognostic scoring system in order to fully understand the correlation between these two modification patterns and GC prognosis.Despite substantial studies on RNA modification, clinical evidence on the correlation between RNA modification and the development of cancer is insufficient. The analysis of several transcript isoforms produced from patterns of APA‐RNA and m7G‐RNA modifications. We detected two unique patterns of RNA modification and discovered that they were associated significantly with the cell cycle, KRAS, TGF‐β, and other signaling pathways related to cancer. Following that, we developed an RNA modification model (RMscore) that allowed us to measure the effectiveness of various alteration patterns in each GC patient. We discovered that the distribution of the RMscore was comparable with the ACRG molecular subtype, implying that this scoring method based on APA‐RNA and m7G RNA modifications plays a major role in determining GC prognosis.The transcriptome data from 2412 GC samples were integrated into this investigation to conduct a comprehensive analysis of the biological patterns resulting from APA‐RNA and m22.1https://www.ncbi.nlm.nih.gov/geo/) for this study: GSSE100935, GSE15459, GSE26253, GSE26942, GSE34942, GSE35809, GSE51105, GSE54129, GSE57303, GSE62254, and GSE84437 : TCGA‐STAD. Of the total 2412 samples, 2037 were from GEO, and the remaining 375 were from TCGA. All raw data in the GEO database were microarray data processed on Affymetrix and Illumina. The raw data retrieved from the Affymetrix platform were processed utilizing the RMA algorithm of the “affy” package in R for background adjustment and normalization.Figure GSE84437 . One dat2.2The unsupervised clustering algorithm was used to perform a combined assessment on several datasets for the 20 marker genes in order to detect RNA modification patterns and classify samples for further investigation. Clustering consensus is a technique for computing the clustering consensus and evaluating its stability via repeated subsampling and clustering. It produces the clustering numbers that are the most closely related to the data matrix.2.3http://www.gsea‐msigdb.org/gsea/downloads.jsp). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway studies were carried out with the help of the “clusterProfiler” package. A 0.05 (FDR) level of significance was considered to be statistically significant.Unsupervised and nonparametric techniques were used to conduct GSVA to detect variations in the enrichment of pathways and biological processes among RNA modification patterns. GSVA was performed using the “GSVA” package in R.2.4p < 0.05 and |log FC| more than 1 .Following consensus clustering, the samples with different RNA modification patterns were classified into phenotype_A and phenotype_B. We utilized the R package “limma” to detect DEGs associated with phenotypes between the two clusters. This software analyses gene expression alteration using P‐values and t‐statistics obtained using Empirical Bayes estimation in a linear model.2.5p < 0.001). Next, we performed Boruta feature selection using random forest machine learning using the “Boruta” package in R.First, we repeated the consensus clustering on 56 DEGs related to phenotype from the 2037 samples to detect gene clusters associated with GC patients. As a result, we obtained three gene clusters for further analysis. Then, using a univariate Cox regression model, we evaluated the DEGs to determine which genes are related to the GC prognosis. Forty‐one significant genes were identified during this investigation (2.6http://tc3a.org).p < 0.05 and PDUI|log2 FC| > 0.12 were considered as a threshold for statistical significance.APA‐mediated 3' UTR shortening is a characteristic of the majority of cancer cells.p < 0.05 and |log FC| > 1 were considered as a threshold for statistical significance.Generally, the miRNA target sites are located in 3' UTRs.2.7https://www.cancerrxgene.org/) database.For drug sensitivity analysis, we collected the drug response data from the Genomics of Drug Sensitivity in Cancer  curve was achieved with the help of the “pROC” software package. Cox regression analyses, both univariate and multivariate, were carried out with the “survival” package in order to compute the hazard ratios and determine the independent prognostic factors in the study. All statistical tests used were two‐sided, with a p < 0.05 considered statistically significant. All of the statistical results were evaluated utilizing SPSS (version 26.0), R (version 3.6.2), and GraphPad Prism (version 9.0).The Shapiro–Wilk normality analysis was conducted to assess the variables' normality. Continuous variables were compared between two groups utilizing the Mann–Whitney 33.17G‐related genes (METTL4 and WDR4). Further analysis of CNV alteration frequency showed that PABPC1 and CPSF1/4/6 had a high frequency of CNV amplification, whereas WDR4, CPSF3, and CLP1 mainly showed CNV depletion . Then, GSVA was carried out to compare the pathway enrichment and biological process between phenotype_A and phenotype_B utilizing gene sets . In most published papers, hallmark gene sets summarized substantially well‐defined biological processes and showed excellent consistency.p < 0.05). To identify the higher proliferation ability of phenotype_B, we compared MKI67 expression between the two clusters and discovered that phenotype_B had considerably greater MKI67 expression . We also discovered genomic subtypes based on the prognostic genes using the consensus clustering method , which was comparable with the pattern of a better prognosis being observed in patients in phenotype_B.The differential analysis of the two RNA modification patterns revealed their potential biological functions. We identified 566 DEGs with |log FC| > 0.585 and an adjusted e Figure E. Furthee Figure . Except e Figure . To deted Figure A–C. Thred Figure . Additiod Figure . A bette7G‐RNA modification patterns.The above results proved the effectiveness and stability of APA‐RNA and m3.47G‐RNA modification patterns of individual GC patients (Table p < 0.01). Kaplan–Meier study demonstrated that patients with a high RMscore had a considerably better prognosis in comparison with patients with a low RMscore . Furthermore, survival analysis was conducted on patients in the ACRG cohort who had adjuvant chemotherapy to assess the prognostic analytic effect of RMscore in patients who took or did not take adjuvant chemotherapy. We revealed that among both patients who received chemotherapy and those who did not, patients with a high RMscore showed higher overall survival , and the area under the curve (AUC) of the ROC was 0.658 .Following that, we conducted uni and multivariate Cox regression analyses to assess whether the RMscore could be employed as an independent prognostic factor for GC. According to the findings, the RMscore is a strong independent prognostic factor. . Next, we attempted to expand the application of this model to other cancers, such as colorectal cancer (CRC). Kaplan–Meier survival analysis indicated that CRC patients with a low RMscore had a poor prognosis . These results demonstrated that RMscore is an effective and stable model to explain APA‐RNA and m7G‐RNA modifications and can predict the prognosis of patients with gastrointestinal tumors, especially GC.In order to validate the stability of the RMscore model, we applied the RMscore signature established in the ACRG cohort to six external independent GEO or TCGA GC datasets. The findings of the validation examination demonstrated that patients with a higher RMscore had a better prognosw3sis, , and we showed the correlation and significance of 30 drugs with the highest correlation coefficients , M2 macrophages (p = 0.047), and resting dendritic cells (p < 0.001) were considerably lower in a group with the high RMscore than in the group with the low RMscore. On the contrary, the activated memory CD4+ T cells (p < 0.001), M1 macrophages (p = 0.032), activated dendritic cells (p = 0.007), and neutrophils (p = 0.004) in the group with the high RMscore were considerably more than those in the group with the low RMscore . We identified the differential PDUI profile between high RMscore and low RMscore groups and compared the prognosis to determine the association of the length of 3' UTR and overall survival of GC patients . However, consistent with a previous study,Given that APA events are essential for posttranscriptional regulation , we assessed the relationship between RMscore and PDUI. We set PDUI > 0.71  as the cutoff for relatively longer 3' UTR isoforms. The percentage of long 3' UTR genes in the group with a high RMscore was considerably greater than that in the group with a low RMscore . To determine the functional features of miRNA and target genes, we constructed the miRNA‐mRNA network that showed the differentially expressed miRNA and mRNA between the high RMscore and low RMscore groups , the RMscore model failed to show consistent efficacy of immunotherapy in these two cohorts. The underlying relationship between RMscore and immunotherapy awaits follow‐up studies. Finally, our results have important implications for GC RNA modification, and the detailed molecular mechanisms need additional investigation to elucidate deeper relationships.57G‐RNA modifications in GC, and we developed the RMscore model based on the modification patterns in order to investigate the vast regulatory mechanisms by which RNA alterations affect carcinogenesis in greater detail. We established a correlation between the scoring system and clinical outcomes in GC patients and validated the model using numerous external datasets from the TCGA and GEO databases. This investigation has provided novel information about the role of RNA modifications in the development of GC.This work focused on the investigation of APA‐RNA and mQC, YT, and HX: conception and design. QC, ZL, and YT: acquisition of data. QC, SP, and WA: analysis and interpretation of data. QC, ZL, and HX: writing and review of the manuscript. The final version of this work has been read and approved by all of the authors.This research was supported by the National Natural Science Foundation of China (No. 81772549), National Natural Science Foundation of China (No. 81572334).The authors state that they had no commercial or financial affiliations that may be considered a potential conflict of interest when conducting the research.Supplementary Figure 1Click here for additional data file.Supplementary Figure 2Click here for additional data file.Supplementary Figure 3Click here for additional data file.Supplementary Figure 4Click here for additional data file.Supplementary Table 1Click here for additional data file.Supplementary Table 2Click here for additional data file.Supplementary Table 3Click here for additional data file.Supplementary Table 4Click here for additional data file.Supplementary Table 5Click here for additional data file.Supplementary Table 6Click here for additional data file.Supplementary Table 7Click here for additional data file.Supplementary Table 8Click here for additional data file.Supplementary Table 9Click here for additional data file.


Pigeonpea sterility mosaic virus-I (PPSMV-I) and Pigeonpea sterility mosaic virus-II (PPSMV-II) were found to be associated with sterility mosaic disease (SMD) of pigeonpea [Cajanus cajan (L.) Millsp.]. The host range of both these viruses and their vector are narrow, confined to Nicotiana benthamiana identified through mechanical transmission, and to Phaseolus vulgaris cvs. Top Crop, Kintoki, and Bountiful (F: Fabaceae) through mite transmission. A weed host Chrozophora rottleri (F: Euphorbiaceae) was also infected and tested positive for both the viruses in RT-PCR. Among the wild Cajanus species tested, Cajanus platycarpus accessions 15661, 15668, and 15671, and Cajanus scarabaeoides accessions 15683, 15686, and 15922 were infected by both the viruses and mite vector suggesting possible sources of SMD inoculum. Though accession 15666 of C. platycarpus, 15696 of C. scarabaeoides, and 15639 of Cajanus lanceolatus were infected by both the viruses, no mite infestation was observed on them. Phylogenetic analysis of nucleotide sequences of RNA-1 and RNA-2 of PPSMV-I and PPSMV-II isolates in southern India revealed significant divergence especially PPSMV-II, which is closely related to the Fig mosaic virus (FMV) than PPSMV-I. In multilocation testing of pigeonpea genotypes for their broad-based resistance to SMD for two consecutive years, genotypes ICPL-16086 and ICPL-16087 showed resistance reaction (<10% incidence) in all three locations studied. Overall, the present study gives a clear idea about the host range of PPSMV-I and PPSMV-II, their molecular relationship, and sources of resistance. This information is critical for the development of reliable diagnostic tools and improved disease management strategies.Two distinct emaraviruses,  Pigeonpea sterility mosaic virus-I (PPSMV-I) and Pigeonpea sterility mosaic virus-II  in a semi-persistence manner (Sterility mosaic disease (SMD) is one of the important production constraints of pigeonpea cultivation in the Indian subcontinent . SMD is e manner . SMD syme manner .Pigeonpea sterility mosaic virus , a putative RNA virus of negative orientation. Complete genome sequences of PPSMV-I was reported to contain four to five RNA segments (RNA-1 to RNA-5) and PPSMV-II with six RNA segments (RNA-1 to RNA-6) have been published recently  in a glasshouse at 27 ± 1°C with 70–80% relative humidity. The leaf stapling technique (echnique  was usedSterility mosaic disease-infected young leaf tissue was ground in 0.05 M phosphate buffer (1:10 w/v) containing 0.1% β-mercaptoethanol (β-ME) using mortar and pestle on an ice bucket, filtered through a muslin cloth, and inoculated immediately onto a test plant at the two- to three-leaf stage by dusting celite (abrasive) (Sigma-Aldrich) with the forefinger. The inoculated leaves were then slowly rinsed with distilled water and kept in a vector-proof glasshouse at 27 ± 1°C.PPSMV was followed as per the protocol described by PPSMV transmission onto the test plant.Leaf-stapling method of transmission of PPSMV were developed at ICRISAT, Hyderabad, and were used to detect the virus in plant tissues by direct antigen coating (DAC)-ELISA as per the protocol suggested by Polyclonal antibodies to PPSMV-I and PPSMV-II. Random-primed cDNA (2 μl) was added to 5× Taq polymerase buffer  containing MgCl2 to a final concentration of 1 mM, 0.2 mM dNTPs, 0.2 μM of each specific primer, and 1 U of Taq DNA polymerase in a final volume of 25 μl. The PCR mixture tube was incubated by 1 cycle of denaturation at 94°C for 4 min, followed by 35 cycles at 92°C for 30 s, 45–65°C for 30 s, and 72°C for 30 s. The final extension was at 72°C for 7 min. The amplification products were resolved in a 1.2% TBE agarose gel, visualized, and documented by a gel–doc system .Briefly, 100 mg of leaf tissue was ground in liquid nitrogen to a fine powder. Total RNA was extracted using the QIAGEN RNeasy plant mini kit by following the manufacturer’s instructions. The RNA quantity and quality were assessed using a spectrophotometer  and stored in a refrigerator at −20°C. RT-PCR was performed as per the protocol suggested by Nicotiana species obtained from the Central Tobacco Research Institute (CTRI), Rajamahendravaram, Andhra Pradesh, India, were used in the study along with five herbaceous plants, such as two pigeonpea cultivars (ICP-8863 and ICP-2376), Phaseolus vulgaris, Vigna unguiculata c-152, and Chenopodium album, and were grown in 8-inch pots and inoculated mechanically with SMD-infected sap and kept inside a glasshouse at 27 ± 1°C. In another set, seeds of 24 accessions of 12 Cajanus species obtained from the gene bank of ICRISAT, India, were scarified by slicing the seed coat with a scalpel blade, treated with thiram at 30 mg/10 g of seeds, and were sown in 8-inch pots along with 16 cultivated species intercropped with pigeonpea and 46 weed species raised to the two- to three-leaf stage and inoculated by leaf stapling method and kept in the glasshouse at 27 ± 1°C with 70–80% relative humidity. The test plants were monitored regularly for symptom appearance and tested for the infection of PPSMV by DAC-ELISA. The ELISA-positive samples were tested further for the infection of PPSMV-I and/or PPSMV-II using oligonucleotide primers corresponding to the RNA-3 segment through RT-PCR.Seeds of 11 Pigeonpea leaves exhibiting typical SMD symptoms were collected during a roving survey conducted in 2017/2018 from different geographical locations covering Andhra Pradesh, Karnataka, Tamil Nadu, and Telangana states in southern India. The collected samples were placed in ziplock plastic bags and transported in cold packs to ICRISAT, Hyderabad, snap frozen in liquid nitrogen, and stored in a −80°C freezer.PPSMV-I and PPSMV-II. Amplicons were purified and sequenced by Sanger’s dideoxy chain-termination method [ABI 3730 (48 capillaries) electrophoresis]. Later, the nucleotide homology searches were done with the BLASTN sequence analysis of the NCBI1. Multiple alignments were performed using MUSCLE , were obtained from the Pigeonpea Breeding Unit, ICRISAT, Patancheru, India. All these genotypes were evaluated for their reaction to SMD at three geographical locations, such as Bengaluru , Coimbatore , and Patancheru , in southern India for two consecutive years (rainy season 2017/2018 and 2018/2019). Plants were raised to the two- to three-leaf stage and inoculated by following the leaf stapling method as described earlier.The test genotypes were regularly monitored for the symptom expression, SMD incidence was recorded, and percent disease incidence was calculated using the formula:Based on the SMD incidence, test genotypes were categorized as resistant (≤10.0% incidence), moderately resistant (10.1–20.0% incidence), susceptible (20.1–40.0% incidence), and highly susceptible (>40.0% incidence) .Nicotiana species (Nicotiana benthamiana was found positive for PPSMV infection. Symptoms appeared after 35–40 days of post-inoculation (dpi) as chlorotic spots, deformation of leaves, and stunted growth, while in the advanced stage (65–70 dpi), the symptoms were systemic and appeared on young leaves as yellow mosaic and crinkled leaves  (Of the 11  species  and fived leaves . HoweverPSMV-II) .Phaseolus vulgaris cvs. Top Crop, Bountiful, and Kintoki (F: Fabaceae) were infected with PPSMV but not supported for mite multiplication. The symptoms appeared as stunting, reduced leaf size, and mild crinkling. A weed host, Chrozophora rottleri (F: Euphorbiaceae) was also tested positive for PPSMV infection; however, there were no visual symptoms of virus infection or mites. A member of Fabaceae, Macroptilium atropurpureum, was found infested with a few mites but tested negative for PPSMV. Similar to the Nicotiana species, all the ELISA-positive samples when tested in RT-PCR were found infected with both the viruses  also clustered with PPSMV-II isolates, whereas the RNA-1 of PPSMV-I isolates Gulbarga BRS1117 and Bengaluru BRS1117 clustered together, and the isolates Tirupati BRS1117, Vamban BRS1117, Patancheru BRS1117, and Tandur BRS1117 clustered in another subcluster. Similarly, distinct clusters were formed when the RNA-2 sequences of PPSMV-I and PPSMV-II were subjected to the phylogenetic analysis in which the RNA-2 sequence of PPSMV-I isolate Coimbatore BRS1117 and PPSMV-II isolate Gulbarga BRS1117 were distinctly separated from the rest of the isolates. The RNA-2 sequence of FMV is also clustered with PPSMV-II isolates. The RNA-1 and RNA-2 sequences of PPSMV-I and PPSMV-II isolates were phylogenetically closer. However, RNA-1 and RNA-2 sequences of PPSMV-II isolates exhibited a close relationship with FMV than the PPSMV-I.The oligonucleotide primers targeted for RNA-1 and RNA-2 of ct sizes . The nucclusters . The isoScreening of pigeonpea genotypes to SMD in different geographic locations revealed considerable variations in response to SMD infection . SusceptPPSMV-I and PPSMV-II, the molecular relationship between them, and the source of resistance to SMD. The host range of PPSMV is narrow and confined to Nicotiana benthamiana through sap inoculation. The evidence of susceptibility of N. benthamiana to a wide range of plant viruses has been provided by PPSMV onto N. benthamiana and Nicotiana clevelandii by sap inoculation, but not on to the pigeonpea or any herbaceous hosts. Through mite inoculation, both the viruses can be successfully transmitted onto Phaseolus vulgaris cvs. Top Crop, Bountiful, Kintoki (F: Fabaceae) and Chrozophora rottleri (F: Euphorbiaceae), and was confirmed by the reports of Hibiscus panduriformis, as in our study, neither the mite infestation nor PPSMV infection was found in both the field-collected samples as well as in artificially inoculated plants of H. panduriformis. Among the 24 accessions of 12 wild Cajanus species tested for the PPSMV infection, the accessions 15661, 15668, and 15671 of C. platycarpus and 15683, 15686, and 15922 of C. scarabaeoides were positive for both the viruses and supported mite multiplication, confirming earlier reports of Our study described the host range of PPSMV-I and PPSMV-II associated with SMD of pigeonpea showed that these two emaraviruses are widespread across southern India. Analysis of sequence identity among the isolates of PPSMV-I and PPSMV-II indicated the presence of significant sequence variability. The RNA-1 and RNA-2 sequences of PPSMV-II isolates exhibited a close relationship with FMV than with PPSMV-I, and it is convincing and in agreement with the previous reports of PPSMV-II in the Coimbatore and Bengaluru locations. However, when we analyzed samples from different geographical locations in southern India, there is an existence of both the viruses in the Coimbatore and Bengaluru locations. This development of mixed infection over the years is possibly due to the spread of the virus to these locations by its mite vector, A. cajani. In nature, mites are the only means of transfer of SMD causal agent to pigeonpea and not through seed, pollen, or soil  can be found in the article/online.BS conceptualized the work in discussion with AP and HS, executed the work in the field and glasshouse, and wrote the initial draft of the manuscript. VP, KJ, KS, and MS provided necessary inputs in designing the work plan. HR, ER, and LK provided all necessary support in the execution of the work in various locations. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Argonautes are small RNA-binding proteins, with some having small RNA-guided endonuclease (slicer) activity that cleaves target nucleic acids. One cardinal rule that is structurally defined is the inability of slicers to cleave target RNAs when nucleotide mismatches exist between the paired small RNA and the target at the cleavage site. Animal-specific PIWI clade Argonautes associate with PIWI-interacting RNAs (piRNAs) to silence transposable elements in the gonads, and this is essential for fertility. We previously demonstrated that purified endogenous mouse MIWI fails to cleave mismatched targets in vitro. Surprisingly, here we find using knock-in mouse models that target sites with cleavage-site mismatches at the 10th and 11th piRNA nucleotides are precisely sliced in vivo. This is identical to the slicing outcome in knock-in mice where targets are base-paired perfectly with the piRNA. Additionally, we find that pachytene piRNA-guided slicing in both these situations failed to initiate phased piRNA production from the specific target mRNA we studied. Instead, the two slicer cleavage fragments were retained in PIWI proteins as pre-piRNA and 17–19 nt by-product fragments. Our results indicate that PIWI slicing rules established in vitro are not respected in vivo, and that all targets of PIWI slicing are not substrates for piRNA biogenesis. Argonautes are small RNA-binding proteins that function in most organisms and in all cells of multicellular organisms . PIWI prThree mouse PIWI proteins are expressed in the male germline, with MIWI (PIWIL1)  and MILIMouse MIWI is exclusively bound by pachytene piRNAs and its loss leads to male infertility . PachyteOne core function of some of the Argonaute proteins is their small RNA-guided endoribonuclease (slicer) activity . This isIn this study, we sought to experimentally determine the in vivo consequence of pachytene piRNA-guided slicer activity on an endogenous mRNA. Using MIWI slicing rules established in vitro , we desiYthdc2 mRNA  complementary binding site for piR-A in the 3′UTR of Ythdc2 (10xPerf) complementary binding sites that are separated with spacer sequences between them. In vitro slicer assays with immunopurified MIWI complexes isolated from mouse testes previously showed that a target RNA that is perfectly complementary to piR-A is cleaved  or ten (10xBulge) mutated binding sites , all lines were maintained in the laboratory by crossing the homozygous lines. Also, there wasn't any dramatic change in target Ythdc2 mRNA levels . We conclude that presence of multiple binding sites for an abundant pachytene piRNA in the 3′UTR of Ythdc2 did not result in gross fertility defects.Knockout of  meiosis , while c meiosis . Slicing meiosis . Slicer  meiosis , as the  meiosis . HoweverYthdc2 mRNA results in generation of new piRNAs, we prepared total testicular small RNA libraries (20–40 nt). We used P14 homozygous knock-in 1xPerf and 1xBulge mice but did not detect any target-derived small RNAs (data not shown). Since the level of piR-A is higher in later stages of spermatogenesis we next examined adult mouse testes  (Reads with their 5′ ends at position −9 are not in the piRNA size-range (26–30 nt), indicating that these are not mature piRNAs. Instead, they have a fixed size of 45 nt E,F and tine (G1) B,G, whicine (G1) . AlternaThe reads with their 5′ ends at positions −26 and −28 have defined lengths of 17 and 19 nt, respectively F, with t10xBulge knock-in mice revealed the tell-tale signatures of slicer activity (Immunopurified endogenous MIWI fails to slice a target RNA when a 2 nt mismatch (at positions 10 and 11 of the piRNA) exists between the guiding piRNA and the cleavage site . Surprisactivity A–C. Firsactivity C. Secondactivity D. We exaactivity E. This sactivity . This isactivity .10xPerf, the pre-piRNA fragment originating from 10xBulge has a length of 45 nt consistent with its 5′ and 3′ ends being generated by slicer activity from two consecutive bulged piRNA-targeting sites .Ythdc2 gene . Binding sites are perfectly complementary (1xPerf or 10xPerf) or contain two mismatches (1xBulge or 10xBulge) on the 10th and 11th position relative to the guide RNA. To create these reporter mouse lines, the Ythdc2 locus of interest was targeted in single cell mouse embryos of the B6D2F1/J hybrid line .Knock-in mice were created by inserting one or ten complementary binding sites for a piRNA (piR-A) in the 3′ UTR of the Ythdc2. Two ssDNA oligonucleotide templates  containing the required single piRNA binding sites were coinjected. The two multiple binding sites reporter alleles were created by separately coinjecting two pUC57-Kan plasmids containing 10 piR-A binding sites (perfect or bulge) separated by spacers and flanked by homology arms to the Ythdc2 locus of interest . The gRNAs were composed of two parts: one tracrRNA and one crRNA (sequences as above). The tracrRNA is 67 bases long  and is the common part of the guide RNA that binds the Cas9. A mixture of 200 pmol of tracrRNA and crRNA were annealed by incubating 5 min at 95°C in 1xTE buffer, pH 7.5  and cooling down at room temperature for 30 min. The final volume was 10 µL. Annealed gRNAs were stored at −20°C and then used for mouse single cell embryo injection.Targeting of the locus was achieved by injecting two different guide RNAs (gRNAs from Integrated DNA Technologies) complexed with the Cas9 protein. The first gRNA (CAUAACGCUCUUCCAUAGCCGUUUUAGAGCUAUGCU) targeted the reverse strand at position chr18: 44887792. The second gRNA (GUGCAUUCUGUAGCUACAACGUUUUAGAGCUAUGCU) targeted the reverse strand at position chr18: 44887815. Combination of these two gRNAs and Cas9 endonuclease created a deletion of 23 bp in the last exon (exon 30) of The single cell embryo injection mix was prepared right before the injection. Cas9 protein at 30 ng/µL final concentration  was mixed with the annealed gRNAs at 0.6 pmol/µL final concentration. Template DNA was also added to the mix at 20 ng/µL final concentration and the volume was adjusted to 100 µL with TE buffer (pH 7.5). Complex formation was achieved by incubating the mix 10 min at room temperature. Finally, the mix was centrifuged at 13,000 rpm for 5 min at 4°C, and 50 µL of supernatant were collected and placed on ice.Ythdc2 gene were used to genotype the knock-in alleles. The expected PCR product sizes were 281 bp for WT, 320 bp for 1xPerf/1xBulge and 716 bp for 10xPerf/10xBulge. PCR reactions were run with Phire Green Hot Start II PCR Master Mix (Thermo) under the following conditions: 98°C for 30 sec, 35 cycles of , 72°C for 60 sec, and lastly at 4°C to hold the reaction.Primers  flanking the targeted insertion region in the mouse Immunoprecipitation from adult mouse testes was conducted as previously reported using anti-MILI and anti-MIWI rabbit polyclonal antibodies . MILI- apiRNA libraries were prepared with NEBNext Multiplex Small RNA Library Prep Set for Illumina  and sequenced at EMBL Genomic Core Facilities in Heidelberg using the NextSeq 500 platform (Illumina).Total RNA extracted from adult whole testes was used as starting material for the testicular transcriptome sequencing. The Stranded Total RNA Ribo-Zero Plus kit from Illumina was used for the library preparation with 500 ng of total RNA as input. Library molarity and quality were assessed with the Qubit and Tapestation (DNA High sensitivity chip). Libraries were sequenced on a NovaSeq 6000 Illumina sequencer for SR100 reads .Reads were sorted into individual libraries based on the barcodes, and the 3′ adapter sequences were clipped from the reads using cutadapt  with onlYthdc2 transcript sequence containing individual reporters: 1xPerf, 10xPerf, 1xBulge and 10xBulge . We calculated 5′ end and body coverages of the reads produced from individual reporters as well as coverages targeting the reporters. Counts of reads were divided by number of mapped sites and normalized to library sizes (rpm—reads per million). To characterize the overall production of piRNAs from the ten sites of 10xPerf and 10xBulge reporters, we also created the metaplots where we summarized the counts of piRNAs produced from individual positions of all ten target sites. The positions refer to the distance from the 5′ end of targeting piR-A piRNA. Therefore, nucleotide at position 0 refers to the nucleotide which pairs with the first nucleotide of piR-A. The position −9 refers to the nucleotide pairing with 10th piR-A nucleotide and is the position of the 5′ end of secondary piRNAs created by MILI or MIWI cleavage.To see whether any piRNAs are produced from the reporters, the reads were mapped by bowtie  (with pahttps://github.com/kevinblighe/EnhancedVolcano). To visualize expression of dysregulated genes across spermatogenic populations (NCBI: PRJNA317251), we calculated the z-scores of log2 expression (reads per million) for each of those genes and plotted the boxplots for individual samples. Similarly, the heatmap of z-scores was plotted using pheatmap 1.0.12.The reads from long RNA-seq libraries were mapped to the mouse genome (GRCm39 - Ensembl release 104) using salmon  salmon  and BiocThe sequencing data generated in the study are deposited with the Gene Expression Omnibus (GEO: GSE219200).Supplemental material is available for this article.


The ventromedial frontal lobe (VMF) is important for decision-making, but the precise causal role of the VMF in the decision process has not been fully established. Previous studies have suggested that individuals with VMF damage violate transitivity, a hallmark axiom of rational decisions. However, these prior studies cannot properly distinguish whether individuals with VMF damage are truly prone to choosing irrationally from whether their preferences are simply more variable. We had individuals with focal VMF damage, individuals with other frontal damage, and healthy controls make repeated choices across three categories—artworks, chocolate bar brands, and gambles. Using proper tests of transitivity, we find that, in our study, individuals with VMF damage make rational decisions consistent with transitive preferences, even though they exhibit greater variability in their preferences. That is, the VMF is necessary for having strong and reliable preferences, but not for being a rational decision maker. VMF damage affects the variability with which value is assessed, but not the consistency with which value is sought. The ventromedial frontal lobes (VMF) contribute to encoding of value. Here the authors show that individuals with VMF damage have less stable, but fundamentally transitive preferences, suggesting that valuation does not solely rely on the VMF. An example of transitivity is the following: If you prefer to listen to Adele (A) over Beyoncé (B), and Beyoncé over Celine Dion (C), then you would also prefer Adele (A) over Celine (C). There is a strong argument that preferences ought to be transitive, as an intransitive chooser would get caught in choice cycles that do not advance towards any goal  and that could be exploited . Thus, nearly all normative theories of decision-making are transitive.A central assumption of rational choice theories is that decision-makers compare the subjective value of different options and choose the highest valued option. Satisfying this assumption is equivalent to having transitive preferences5. Correspondingly, lesions to the VMF impair value-based decision-making in a variety of ways10.Given this, one might expect that organisms have internal representations of value that are transitive. Dozens of functional neuroimaging studies in humans and neurophysiological studies in non-human animals have now identified neural activity in the ventromedial frontal lobe (VMF) that scales with subjective value across different categories of goods13. This increase in cyclical choices after VMF damage, however, is consistent with two very different possibilities regarding the necessary role of VMF, and putative value signals in VMF, in rational choice.However, the precise role of the VMF in rational choice is still unclear. Intriguingly, several previous studies have shown that individuals with VMF damage make more cyclical choices  than healthy controls or individuals with damage elsewhere in the frontal lobeThe first possibility is that the preferences of individuals with VMF damage are fundamentally intransitive and not self-consistent. In this case, in the example above, the individual with VMF damage would consistently and reliably choose C over A. This could occur if individuals with VMF damage choose according to stimulus-response associations or rules that lack any higher order transitive structure, rather than according to any set of underlying preferences. If this possibility were true, the proper conclusion would be that an intact VMF is necessary for the human brain to assess value at all, or to use value to make decisions; i.e., that an intact VMF is necessary for transitive preferences and rational choice.The second possibility is that the preferences of individuals with VMF damage are transitive and self-consistent, but more variable. In this case, the individual in the example above might prefer A over C on average, but less decisively than others. Thus, if asked again later, this individual would have a greater chance of changing their mind and now choosing C over A. This change of mind could occur if individuals with VMF damage choose according to underlying values, but the assessment of those values was more variable across time. If this possibility were true, the proper conclusion would be that an intact VMF promotes the stability and reduces the variability of valuations across time; but an intact VMF is not necessary for transitive preferences and rational choice.14. Importantly, these authors recognize that behavior in experiments is probabilistic, and therefore testing axioms of rational choice like transitivity requires recasting these axioms in probabilistic terms17. Critically, counting the number of observed choice cycles , as done in previous studies, is unrelated to the degree to which choices violate a probabilistic model of transitivity. Put differently, counting choice cycles does not help to disentangle whether one has fundamentally intransitive versus variable preferences. We use these recently developed tests of a probabilistic model of transitivity to determine whether the preferences of individuals with VMF damage are more variable or fundamentally intransitive. If the choices of individuals with VMF damage do not satisfy these tests, we would have evidence that their preferences are fundamentally intransitive. Alternatively, if their choices do satisfy such tests, it would suggest that previous findings of more choice cycles are due to variability in preference . A definitive discrimination between these two possibilities will identify more precisely the necessary role of VMF, and putative value signals in VMF, in value-based decision-making.Distinguishing between preferences that are fundamentally intransitive and preferences that are simply more variable is a deep problem in testing theories of rational choice that has only recently been solved for the example of transitivity by Regenwetter and colleaguesWe tested thirteen individuals with VMF damage, and for comparison, ten individuals with damage to frontal lobe  other than VMF , and twenty age and education-matched healthy controls . Figure Participants made binary preference decisions in three categories: artworks, chocolate bar brands, and monetary gambles. Each category had two sets of options. Set A, used for probabilistic tests of transitivity, contained 5 items that were used to construct 10 binary choices that were each repeated 15 times throughout the experiment. Set B, used to test for choice cycles, contained 10 or 11 items  that were used to construct 45 or 55 binary choices that were each presented once. Choices using set A and set B items were intermingled in each block. All items in the artwork and chocolate bar brand sets were normed to be as close in value as possible, and all gambles were of equal expected value (see “Methods” for details).Our central question is whether the choices of individuals with VMF damage would violate a probabilistic model of transitivity. If they do, this would show that the preferences of individuals with VMF damage are fundamentally intransitive; if they do not, this would suggest that their preferences might be more variable, but are not intransitive.To answer this question, we examined the subset of choices in our experiment, set A, which involves 15 repetitions each of 10 different binary choices in each of the three categories. In each category, we can calculate 10 choice percentages, one for each of the possible pairings of five items. Table 19 assumes that every choice is made according to a preference ordering, but that the preference ordering governing a specific choice is drawn randomly from a mixture of all possible preference orderings. Regenwetter and colleagues14 show that these assumptions place a constraint on the observable choice percentages called the triangle inequalities (see “Methods”) and develop novel statistical methods for determining when violations of the triangle inequalities are unlikely to be due to random sampling. For five stimuli and thus ten pairwise choice percentages, the triangle inequalities fully characterize the mixture model and impose rather restrictive constraints; only 5% of the sample space satisfies the triangle inequalities.These data are sufficient to evaluate whether the choices each participant made are consistent with a probabilistic model of transitive choice called the mixture model. The mixture modelAcross the individuals tested, most choices—including all of those from individuals with VMF damage—were consistent with this probabilistic model of transitivity . Specifically, we modelled the decision process as resulting from a decision variable (DV), which starts at an initial value (int) that is constant across trials, and then, after a period of non-decision time (ndt), increases linearly across time with a slope d*vα, where d is the drift rate, v is the rank difference between the two items, and α is an exponent accounting for potential non-linearities in the effect of rank difference. At each time step, there is also Gaussian variability with a standard deviation of ε added to the DV, which is the key parameter of interest in our case  in each category to a drift diffusion model (DDM)ε . Specifically, the VMF group  had significantly higher ε than HC  and FC  . No other parameters differed between the three groups , but no main effect of category (p = 0.71) nor a group x category interaction (p = 0.71). Then, we conducted a follow-up planned comparison between groups, combined across all three categories. Similar to previous studies, our VMF group  made more cyclical choices than the HC group . The percentage of cyclical choices we observed were also roughly similar to those previously reported for these two groups12.We replicated the finding that individuals with VMF damage have more choice cycles than HC individuals Fig. . We firsp = 0.02) and the difference between the VMF and FC groups was not significant . This increase in cyclical choices in the FC group was unexpected, and we consider possible interpretations of this result in the discussion.However, we did not replicate that this increase in cyclical choices is selective to VMF damage in the frontal lobe. Unlike previous studies, our FC group  also made more cyclical choices than the HC group  = 2.04, p = 0.03; VMF8: t(19) = 3.28, p = 0.003, VMF10: t(19) = 3.13, p = 0.003; VMF13: t(19) = 3.13, p = 0.003). These differences remained significant in the latter three individuals after correcting for multiple comparisons using FDR . As described in the Supplemental Materials, we did not find that these three individuals differed from others in the VMF group in lesion location, lesion volume, or on any demographic variables. In contrast to the VMF group, none of the individuals in the FC group made significantly more cyclical choices than HCs .In addition to testing whether individuals with VMF damage exhibited more choice cycles as a group, we also examined the number of cyclical choices at the individual level. We did this to establish that our conclusions applied not only at the group level for individuals with VMF damage as whole, but also at the individual level for at least some specific individuals within that group . To do this, we considered each individual with a VMF or other frontal lesion as a single case, and compared their percentage of choice cycles  against HCs. We made this comparison using one-tailed case-control p = 0.02), but was not significant across all subjects (rho(38) = 0.13, p = 0.42).Finally, given that VMF subjects exhibited both increased choice cycles compared to HC subjects and a higher variability parameter in the DDM, we examined the correlation between these two measures. This correlation was significant in the VMF group (Spearman rho(10) = 0.67, 13; these studies cannot distinguish between two possibilities, with drastically different implications for the function of the VMF. One possibility is that individuals with VMF damage have preferences that are fundamentally intransitive. A second possibility is that individuals with VMF damage have preferences that are more variable, yet still fundamentally transitive. Here we provide a clear test between these two possibilities by evaluating whether the choices of individuals with VMF damage satisfy a probabilistic model of transitivity. This model assumes that choices are always generated according to a transitive preference ordering, but allows the specific ordering to vary from choice to choice. Choosers with variable preferences fit this model, but choosers with intransitive preferences do not. We find unambiguous evidence that individuals with VMF damage have fundamentally transitive preferences, even though their preferences are more variable, as all individuals with VMF damage make choices in all domains that are consistent with a probabilistic model of transitivity.Past demonstrations that individuals with VMF damage more frequently make cyclical choices 12, “values are unstable, fluctuating from trial to trial in those with VMF damage.” We provide further support for this claim by fitting a DDM20 to each individual’s choices. In this model, the VMF group had significantly higher variability than healthy individuals or those with frontal damage outside the VMF. Importantly, the VMF group did not differ from others in any other parameter of the DDM, showing that preference variability was the only dimension affected by VMF damage.None of the individuals with VMF damage in our study violated the mixture model, even though their decisions exhibited more choice cycles than healthy controls. We interpret this pattern of results to mean that individuals with VMF damage have preferences that are more variable. That is, as suggested by25 and that have shown that disruptions of VMF cause higher choice variability27. Many of these studies have modelled variability as arising from randomness in underlying values, such as in the DDM28, and thus have assumed a probabilistic model of transitivity. However, such valuation models are not the only potential account of choice30. Our study takes a step beyond previous work by demonstrating that such valuation models are merited in our data, using a test of a particular probabilistic model of transitivity31, the mixture model. The choices of all our subjects in the artworks category, and most of our subjects in the brands and gambles categories, were consistent with the mixture model. Combined with previous work testing this model14, this suggests that human choices in a wide variety of domains are consistent with transitivity.These results, particularly that individuals with VMF damage have higher decision variability in the DDM, are broadly consistent with previous studies that have linked choice variability to variability in neural value signals in VMF32. This view would predict that individuals with VMF damage would only be able to choose in a non-value-based manner, for example, according to rules or heuristics. Rules and heuristics can approximate transitive preference orderings under some conditions33, but are not generally guaranteed to do so15. In contrast, these results are easier to reconcile with a framework in which valuation and value-based choice are distributed processes, to which multiple regions of the brain contribute in some respect34. This framework would predict that other regions can compensate for damage to the VMF, so that VMF damage does not fundamentally abolish the transitivity of preferences. Because making transitive choices that maximize value is incredibly important to the survival of an organism, it would make sense that valuation is a highly conserved process that is robust to damage to one part of the cortex.These results are difficult to reconcile with the view that the VMF is the only critical substrate for value-based choice2. Whether striatal damage affects the variability of value-based decisions has not been studied to our knowledge, though striatal damage does impair value-based learning35. Similar to VMF damage, damage to the hippocampus also results in more variable or inconsistent decisions37. The hippocampus has been proposed to support deliberation about value, perhaps by retrieving evidence about value from memory38. The VMF is anatomically connected to, and functionally interacts with, both striatum and hippocampus, and so either or both networks may contribute to value-based choice40. Future studies could use functional brain imaging to more directly test hypotheses about brain networks that may compensate in individuals with VMF damage.On this distributed view, there are several structures that may interact with the VMF to support value-based choice. Like the VMF, neural activity in the striatum reliably scales with subjective value across dozens of functional neuroimaging studies41 and schema formation43.Future studies could further investigate exactly how the VMF supports the stability and reduces the variability of preferences. One possibility is that VMF contributes to the computation of subjective value. If the subjective value is computed through the interaction of several brain regions, the loss of VMF may make this computation more variable and less reliable. An alternative possibility, though, is that the VMF contributes to the same preference ordering being repeated reliably, without contributing to valuation per se. For example, individuals might use episodic memories of their previous choices  to guide their decisions, or a representation of the context of the experiment may activate a specific set of preferences, as in a schematic network. Previous work has shown VMF involvement in both episodic memory processes12. One difference between our study and previous ones is that ours included the choice category of gambles. As the dorsomedial frontal, dorsolateral frontal, and insular cortices have been previously implicated in decisions about risk45, the inclusion of choices between gambles could account for the discrepancy in results. Future studies should examine the generality and replicability of our findings regarding other frontal regions. We hesitate to draw strong conclusions about the role of these other frontal regions in preference variability based on our results alone. First, unlike individuals with VMF damage, the frontal controls did not differ from healthy controls in decision variability, or any other parameter, when their choices were fit to a DDM. Thus, the differences in the frontal control group were only observed in one of the two choice sets in our experiment (set B), whereas the differences in the VMF group were observed in both choice sets (sets A and B). Second, unlike individuals with VMF damage, the frontal controls only made significantly more cyclical choices at the group level; none of the individuals in the frontal control group made significantly more cyclical choices at the individual level when compared to healthy controls in a case-control test. Nonetheless, to the extent that damage to frontal regions outside the VMF also increases preference variability, this would further support the view that valuation is a distributed process in the brain.Unexpectedly, we also found that the frontal control group made more cyclical choices than healthy controls. This finding is inconsistent with previous studies, where an increase in cyclical choices was specific to VMF damage in the frontal lobe47 and potentially explains why studies using similar decision-making paradigms in individuals with VMF damage can yield different results48. Our findings further characterize the necessary role the VMF plays in value-based decision-making. Specifically, though each choice still reflects a subjective preference ordering after VMF damage, an intact VMF is necessary for these preference orderings to remain stable and reliable across time and contexts.In conclusion, we provided a clear-cut test of whether VMF is necessary for transitive preferences and rational choice and found that individuals with VMF damage have preferences that are more variable but, without exception, fundamentally transitive. This result clarifies how apparently erratic choices manifest after damage to the VMF49. Individuals were eligible to participate if they had a lesion primarily affecting the frontal lobes. One individual was excluded due to incomplete data collection . Fourteen females and 9 males were included in the final sample. Participants were tested a minimum of 5 months after injury .Fourteen individuals with focal damage to the frontal lobes were recruited from the Focal Lesion Database (FoLD) at the University of Pennsylvania, and ten were recruited from the Cognitive Neuroscience Research Registry at McGill UniversityParticipants were divided into two groups a priori based on location of damage, assessed with MR or computed tomography images by a neurologist blind to task performance. The VMF group consisted of individuals who sustained damage to the VMF, which is defined as the medial wall below the genu of the corpus callosum, and the orbitofrontal cortices. The FC group consisted of individuals who sustained damage to the frontal lobe sparing the VMF, which includes damage to dorsolateral, dorsomedial, and insular cortices. Lesions were drawn on a common space [Montreal Neurological Institute (MNI) brain] by neurologists at the research sites blind to task performance. The overlap images for the groups are presented in Fig. N = 14) and McGill University (N = 6), including 15 females and 5 males . Participants were tested at the Hospital of the University of Pennsylvania, at the MNI, or at their own home in the greater Philadelphia or Montreal area. Participants saw stimuli on a laptop monitor and responded using the 1 and 0 keys of the keyboard.Choice items consisted of images of artwork, chocolate bars that differed in brand, and gambles presented as pie charts. There were two sets of items: 5 items from each of the categories  that were used in repeated choices that allow a probabilistic test of transitivity (set A); and 10–11 items from each of the categories  used in non-repeated choices that allow testing for choice cycles (set B). Choices constructed using set A and set B stimuli were intermingled in each block. For both the artwork and chocolate bar brand categories, we designed item sets in which the items were normed to be close in preference. The gambles in the gamble category were all of equal expected value .Participants completed a binary forced choice task. On each trial, participants first saw a central fixation point for 1 s, then a screen with two choice items (placed to the left and the right of the center). Participants indicated which item they preferred, by pressing buttons for left or right. Participants had as much time as they needed to make their selection. Following their selection, there was an inter-trial interval of 1 s where a black screen was presented.p-values not equal to 1 in the QTEST 2.1 results50. For set B stimuli, participants faced all possible pairings of either 10 (for brands) or 11 (for artworks and gambles) items, constituting 45 or 55 pairs in total, respectively, and each pair was presented once. Therefore, there were 195 (for brands) or 205 (for artworks and gambles) total choices in each category across the entire experiment.For set A stimuli, participants faced all possible pairings of 5 items, constituting 10 pairs, and each pair was repeated 15 times. The burden of prolonged testing, particularly on our older subjects, many of whom have brain injuries, limited us to using 15 repetitions per choice per person. This is smaller than the common rule of thumb of 20 for asymptotic tests of binomials. This smaller sample size may slightly bias those p = 0.12).Choice trials were presented in blocks, in which participants made choices between items within a single category . There were five blocks of choices for each category, containing 39 (for brands) or 41 (for artworks and gambles) trials each. Each block contained 30 choices composed from set A and 9 or 11 choices composed from set B. Choices from set A and set B were intermingled with each other within a block, with the set B choices inserted into a block of A choices in positions randomly selected from a uniform distribution. We took several steps to reduce any potential memory effects for repeated set A choices. We designed the sequence of trials so that: (1) the same pairing was not repeated within a minimum of 3 trials; (2) the same item rarely appeared on immediately adjacent trials (no more than 9 times throughout the entire experiment); and (3) when the same pairing was repeated the choices immediately preceding and following that pairing differed from its previous occurrence . Furthermore, the side on which stimuli were presented was counterbalanced across repetitions. Finally, we divided the experiment into two sessions, held on separate days for every subject except two (due to scheduling constraints). The two sessions were held on average 8.09 (sd = 11.73) days apart . We did not observe a significant correlation between the total number of choice cycles and days between the two sessions (r(41) = 0.24, 14. The mixture model states that a person’s response comes from a probability distribution over all possible strict linear orderings of the items. Thus, preferences are transitive, but one’s transitive state at any given time can vary. The probability of a person choosing one item (X) over another (Y) is the sum of all the preference states in which X is preferred to Y. In a two alternative forced choice task, this probability is constrained by the triangle inequalities. For every distinct X, Y, and Z in a choice set:xy denotes the probability of choosing X over Y, etc. For up to 5 options in a two alternative forced choice task, satisfying the triangle inequalities is necessary and sufficient for a set of choices to be consistent with the mixture model.All data were analyzed with MATLAB (Mathworks). We used the set A choices to perform tests of a probabilistic model of transitivity. We first obtained the choice percentages  for each of the 10 choice pairs  in each category. We then tested the mixture model of preference described by Regenwetter and colleagues52 software to determine whether these violations were unlikely to be due to random sampling. QTEST 2.1 uses maximum likelihood estimation to find the goodness of fit of the data at each vertex in the linear ordering polytope defined by the triangle inequalities, using a chi-bar-square distribution with simulated weights51. A p < 0.05 was taken as evidence that a subject’s choices in that category were inconsistent with the mixture model of preference.For choice percentages that did not satisfy the triangle inequalities, we used the QTEST 2.120 to the choices and RTs from all set A choices for every subject and category in our experiment. We modeled the decision process as a decision variable (DV) that increased linearly with a slope d*vα, where d was the drift rate, v was the value difference between the items , and α was an exponent accounting for potential non-linearities in the effect of rank difference. We also assumed that at each time step there is Gaussian variability added to the DV, with a standard deviation of ε. We assumed 10 ms time steps. We also assumed there is a non-decision time (ndt) before accumulation begins, and an initial value (int) of the DV that is constant across trials. Choices are made when the DV crosses a threshold.We fit a DDMd, α, ε, int, and ndt. Note that the threshold was a fixed parameter across subjects, as one of the threshold, d, or ε must be fixed for the other two parameters to be estimable. We chose to fix threshold after a model-comparison process showed that option to provide the best model fits. Threshold was held constant at (+/−) 0.15. Values for d were sampled between 0 and 1, for ε were sampled between 0 and 1, for α were sampled between 0 and 3, for int were sampled between the threshold bounds, and for ndt were sampled between 0 and the minimum RT minus 10 ms for that subject.Thus, there are five free parameters: Tcorrect or Tincorrect, where “correct” was defined as choosing the option of higher rank) across all time steps. For each trial, we then calculated the joint likelihood of the subject’s choice at the time which they made that choice , by taking the derivative of this cumulative probability at the timestep of the subject’s choice (every 10 ms to the maximum RT for the subject). The model was then fit using the MATLAB function fmincon, where the cost function was defined as the sum of the negative log likelihoods of the instantaneous probabilities of the subject’s choices and RTs in all trials. The fitting procedure was repeated 10 times for each subject, with each iteration varying in randomly sampled starting values for the free parameters as specified above; the parameters with the lowest log likelihood out of the 10 was taken for that subject. The model was fit individually to each of the three categories  for each subject.To fit these free parameters, we first calculated the cumulative probability that the DV crossed the threshold for the subject’s choice  to provide a more conservative definition of cyclical choices. We used a 2-way mixed measures ANOVA of the number of choice cycles made by each participant, with group as a between-subjects factor and item category as a within-subject factor, followed by one-tailed Wilcoxon ranked sum post hoc pairwise tests when testing for effects between VMF and HC .We used the set B choices to directly replicate previous studies that counted the number of choice cycles. We first determined the preference ordering within each category for each subject. The 10 or 11 options within each category were ranked according to how many times each was chosen by that subject. Then, for each trial, a choice was counted as cyclical if a lower-ranked item was chosen over a higher-ranked item. FollowingFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) genes, respectively. ScriptTaq COVID PCR assay was able to detect up to 100 copies per reaction of the viral RdRP and N genes. The test demonstrated an overall agreement of 95.4%, a positive percent agreement (PPA) of 90.2%, and a negative percent agreement (NPA) of 100.0% when compared with two commercially available kits. ScriptTaq COVID PCR diagnostic test is a specific, sensitive, and low-cost alternative for low-resource settings.The COVID-19 pandemic necessitated an extensive testing for active SARS-CoV-2 infection. However, securing affordable diagnostic tests is a struggle for low-resource settings. We report herein the development and validation of an in-house multiplex real-time RT-PCR diagnostic test for the detection of active COVID-19 infection (ScriptTaq COVID PCR). Furthermore, we describe two methods for RNA extraction using either an in-house silica column or silica-coated magnetic beads to replace commercial RNA extraction kits. Different buffer formulations for silica column and silica-coated magnetic beads were tested and used for RNA isolation. Taq polymerase enzyme and thermostable reverse transcriptase enzyme were purified from bacterial clones. Primers/probes sequences published by the WHO and CDC were used for the qualitative detection of the  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus and is the causative agent for coronavirus disease 2019 (COVID-19). The early identification and isolation of infected individuals is pivotal for COVID-19 pandemic control. Currently, diagnosis of acute SARS-CoV-2 infection relies on either viral RNA or viral antigens’ detection. Viral RNA tests detect nucleic acid sequences specific to SARS-CoV-2 by real-time reverse transcriptase polymerase chain reaction (rRT-PCR), which is currently considered the most sensitive and specific SARS-CoV-2 test. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) tests have been proposed as a low-cost alternative to detect SARS-CoV-2 , but hav® Probe 1- Step RT-qPCR System. The WHO test targets viral NSP12 (RdRP) and E genes, while the CDC test targets viral N genes; both protocols require amplifying each gene separately to prevent false positives. Hence, more reagents are consumed to diagnose each COVID-19 case. In this regard, formulating an in-house multiplex rRT-PCR master mix to detect at least two SARS-CoV-2 genes without false positive results represents a challenge and is necessary to reduce costs and overcome the shortage of reagents. Similarly, RNA extraction kits can also become a major bottleneck owing to limited supplies. In this respect, we report herein the development and validation of an in-house multiplex real-time RT-PCR diagnostic test for the detection of active COVID-19 infection (ScriptTaq COVID PCR). Furthermore, we describe two methods for RNA extraction using either in-house silica column or silica-coated magnetic beads, to replace commercial RNA extraction kits.COVID-19 rRT-PCR kits are costly, thus making mass screening a burden for low-resource settings. Moreover, kit manufacturing can be hindered by deficiencies in required enzymes. Commonly available tests include COVID-19 rRT-PCR commercial kits and rRT-PCR COVID-19 assays developed by the World Health Organization (WHO) or Centers for Disease Control and Prevention (CDC) ,7, whichBacterial expression vectors pUC57-2019-nCoV-PC: N and pUC57-2019-nCoV-PC: RdRP were purchased from Lucerna-Chem AG , while RPP30 (NM_001104546) Human Tagged ORF Clone was obtained from Origene . Following induction, the bacteria were processed for RNA extraction by several buffers to optimize the RNA extraction method. Two RNA purification methods were compared in this study: The silica-column-based method and magnetic-beads silica-coated based method. For the magnetic-bead silica-coated based method, different magnetic systems were evaluated. Biocomma magnetic beads  were used. SpiralPipet (M0022-S) Mix magnetic bead handling system, DynaMagTM-2 Magnet , and Bio-Nobile QuickPick were compared in the magnetic-beads-based method. Invitrogen PureLink RNA mini kit  and QuickPick™ SML total RNA kit  were used as reference methods.The in-house RNA isolation kits established tested two lysis buffers and two wash buffers that would allow the collection of RNA only or combined RNA and DNA. Guanidine HCl, Guanidine Isothiocyanate, Tris, Tris-HCl, Nonidet P40, TWEEN 20, TCEP, Sodium Citrate, N-Lauroyl Sarcosine, Antifoam A, EDTA, ethanol, sodium acetate, NaCl, and nuclease-free water were all purchased from Sigma-Aldrich, Burlington, MA, USA. In-house magnetic beads and silica-column-based RNA isolation methods included the following buffers: Lysis Buffer I  or Lysis Buffer II , Wash Buffer I (3 M sodium acetate pH 5.2), and Wash Buffer II . The in-house magnetic-beads-based nucleic acid (RNA/DNA) isolation method included the lysis buffer described above and Wash Buffer I  and Wash Buffer II . The elution buffer in all methods was nuclease-free water.Lysis Buffer (300 μL) is added to the sample (200 μL) followed by 2.5 μL (40 mg/mL) proteinase K. The sample is vigorously vortexed for 15 s and incubated for 10 min at RT with intermittent vortexing. When Lysis Buffer I is used, an equivalent volume of 100% ethanol (500 μL) is added. This step is bypassed when Lysis Buffer II is used as it contains Ethanol. Then, 10 μL of magnetic beads is added, and the sample is gently vortexed and incubated for 5 min with continuous mixing. Magnetic beads are collected using a magnetic separator and the supernatant is removed. Wash Buffer I (500 μL) is added, and the sample is gently vortexed and incubated for 30 s with continuous mixing. Magnetic beads are then collected and the supernatant is removed. Wash Buffer II (500 μL) is added, and the sample is gently vortexed and incubated for 30 s with continuous mixing. Magnetic beads are then collected using the magnetic separator and the supernatant is removed. Magnetic beads are then air-dried and nucleic acid is eluted with 100 μL of nuclease-free water. Finally, magnetic beads are collected and the supernatant is transferred to a new tube. Nucleic Acid Extraction Using ABNOVA SpiralPipet and Autostage System reagents are added to the Auto Plate when using ABNOVA SpiralPipet and Autostage System, as indicated in g. This is repeated until the entire sample has been processed. Wash Buffer I (700 μL) is added to the 2 mL silica spin cartridge , followed by centrifugation for 15 s at 12,000× g. The spin cartridge is then washed twice with Wash Buffer II (500 μL). The spin cartridge is then centrifuged for 1–2 min at 12,000× g. Finally, RNA is eluted with 30–100 μL RNase-free water. Three sequential elutions of 100 μL each are performed when the RNA yield is expected to be more than 100 μg.Lysis Buffer (1000 μL) is added to the sample (200 μL) followed by vigorous vortexing for 15 s and incubated for 5 min at RT with intermittent vortexing. The lysed sample (700 μL) is then transferred to the spin cartridge and centrifuged for 15 s at 12,000× WHO Coronavirus Charite protocol for COVID-19 has already been published . This meg for 15 min and the pellet is resuspended in 25 mL of buffer A . Resuspended pellets are frozen in liquid nitrogen and thawed at RT twice. Then, 1 mL of lysozyme (100 mg/mL) is added, followed by incubation for 15 min at RT. Then, 25 mL of lysis buffer  is added, followed by incubation for 30 min at 75 °C. The lysis mixture is centrifuged at 16,000× g for 10 min at 4 °C to remove the cell debris. To remove DNA and RNA, 500 µL of the digestive solution  is added and incubated for 5 min at RT. The mixture is incubated for 30 min at 75 °C to denature DNase Ι, followed by centrifugation at 16,000× g at 4 °C for 10 min. Ethanol is added to the supernatant to a final concentration of 55% followed by centrifugation at 16,000× g at 4 °C for 15 min. Finally, the pellet containing Taq Pol is dissolved in 10 mL of storage buffer  and stored at −20 °C. Polymerase activity is determined as described previously - or [32P]-labeled nucleotides employed for the setup of the classical RT assay. The yield for thermally stable reverse transcriptase was on average 65 mg/Liter of LB/Ampicillin Solution, with an activity of 3450 units/mg protein, and with a total of 224,250 units/L of the bacterial culture.Cell lysates are prepared as described above. Reverse transcriptase-producing bacteria are induced by 0.5 mM IPTG overnight and the pellet is resuspended in 25 mL of buffer A . Resuspended pellets are frozen in liquid nitrogen and thawed at RT twice. Then, 1 mL of Lysozyme (100 mg/mL) is added and the mixture is incubated at RT for 15 min. Then, 25 mL of lysis buffer  is added and the lysis mixture is incubated at RT for 30 min. A slurry of pre-swollen TALON4, 1 M betaine, 0.4 mM dNTP, 0.5 mM TCEP, 0.02 U/µL RNase inhibitor, 0.125 U/µL Taq polymerase, 8 U/µL reverse transcriptase); primers/probe for RdRP, N, and RPP30 genes (F: 0.4 µM/ R: 0.4 µM/ Probe: 0.2 µM); and 5 µL nucleic acids processed sample. Tris, KCl, MgSO4, betaine, and TCEP were purchased from Sigma-Aldrich, while RNase inhibitor (RNasin) and dNTP were purchased from Promega . RNA isolated following IPTG induction of transformed E. coli with either pUC57-2019-nCoV-PC: RdRP, pUC57-2019-nCoV-PC: N, or RPP30 clones were used as positive controls. Fluorescence rRT-PCR detection was carried out on Biorad™ CFX96 Real-Time PCR Detection System with CFX Maestro Software (V. 2.3). Cycle parameters were set according to reverse transcription , cDNA pre-denaturation , and PCR cycling for 40 cycles .The primers/probe sequences are listed in p-value < 0.05 was used to assess the significance of all statistical analyses. The results are presented as mean ± standard deviation (SD).Statistical analysis was carried out using SigmaStat software ver. 3.5 . Significant changes in mRNA yields were computed for rRT-PCR results, and analysis was carried out using one-way ANOVA to compare the independent groups followed by (Holm–Sidak method) for pairwise comparisons. One-way ANOVA on Ranks was run followed by Dunn’s test for pairwise comparisons when normality distribution failed. RdRP, N, and RPP30 genes were induced with IPTG to produce corresponding mRNAs. The bacteria following induction were processed for RNA extraction by several methods to optimize the RNA extraction method. Two RNA purification methods were compared in this study: The silica-column-based method and magnetic-beads silica-coated based approach. Tris(2-carboxyethyl) phosphine (TCEP) was utilized in our in-house RNA isolation. By modifications of the wash solution, either RNA or RNA/DNA were isolated in our in-house RNA isolation method. The effect of ethanol concentration on RNA binding to silica columns was determined using Lysis Buffer II. The ethanol concentration of 50% had the maximum yield compared with 25% and 35%  Mix magnetic bead handling system, and Bio-Nobile QuickPick, which were compared in the magnetic-beads-based nucleic acid isolation methods. Utilization of DynaMagTM-2 Magnet resulted in maximum RNA yield  Mix magnetic bead handling system showed the best performance with silica-based spin columns  and Wash Buffer II . Our goal was to develop a method that could isolate RNA/DNA to be used clinically for all DNA and RNA viruses. Treatment of the sample with DNase I would result in the collection of RNA only, similar to QuickPick™ SML total RNA purification kit. The results indicate that RNA/DNA isolation using the above-mentioned washing buffers would result in a high RNA/DNA yield with 10 µL of 50 mg/mL magnetic beads, even when the incubation of samples is carried out at RT . Compariod at RT . RdRP) gene and E gene, markers for SARS-CoV-2, and RPP30 gene by rRT-PCR to monitor nucleic acid extraction efficiency and the presence of PCR inhibitors. On the other hand, the CDC COVID-19 assay can qualitatively detect the N gene for the new SARS-CoV-2 and the RPP30 gene, which can confirm the validity of all test reactions. ScriptTaq COVID PCR assay design and validation strategy chose to exclude the E gene designed by Corman et al. [RPP30 gene used in the CDC and WHO assays  and N genes of SARS-CoV-2 RNA. Alignment analysis demonstrated 100% inclusivity for SARS-CoV-2 primer/probe sets for the RdRP gene and N gene. The alignment results for both genes are shown in No cross-reactivity was detected for the ScriptTaq COVID PCR assay when evaluated by both in silico analysis and by wet testing . The in The clinical performance of ScriptTaq COVID PCR assay  was estaInfection prevention and control measures are critical to combat the COVID-19 pandemic. The current testing utilizes either commercially available kits or the WHO- or CDC-developed COVID-19 assay, which requires commercially available PCR master mixes and RNA isolation kits. Commercially available coronavirus tests are currently available from different companies. However, these kits are expensive and the production of COVID-19 rRT-PCR kits is still slow and limited by shortages in the enzymes utilized in the manufactured kits. COVID-19 rRT-PCR assays also require viral RNA isolation kits and systems such as MagNA Pure Compact Nucleic Acid Isolation Kit I  and NucliSENS easyMAG reagents and accessories , which are also expensive. Thus, the adaptation of the in-house COVID-19 rRT-PCR assays would speed up testing and overcome the resource barrier in many world regions.RNA isolation involves three steps: cell lysis, separation of unwanted products, and RNA elution. RNase activity must be inhibited throughout the procedure . Many diRPP30 gene is used as an internal control for sample collection and nucleic acid isolation method in the WHO and CDC assays. A high NTC associated with the RPP30 gene primers/probes used was observed in both the CDC- and WHO-based assays with in-house multiplex PCR or commercially available PCR master mixes in our laboratory. A new probe for the RPP30 gene had to be redesigned to give an NTC of 0 CT in the multiplex PCR. The WHO assay targets the RdRP and E genes, while the CDC rRT-PCR assay target the SARS-CoV-2 nucleocapsid (N) protein gene and could be complemented by the E gene for screening and confirmation. Beta-coronavirus (β-CoVs or beta-CoVs) is one of four genera  of coronaviruses. The beta-coronaviruses of the most significant importance concerning humans are OC43, and HKU1 of lineage A, SARS-CoV and SARS-CoV-2 of lineage B, and MERS-CoV of lineage C. Alignment analysis using EMBOSS NEEDLE for primers/probes for the E gene indicated high scores for SARS-CoV-2, SARS-CoV Tor2 strain, SARS coronavirus Urbani strain, and human coronavirus OC43 strain and HKU1 of lineage A. Thus, the E gene was excluded from the ScriptTaq COVID PCR assay as beta-coronaviruses could be amplified with the used primers/probes for the E gene.The details of rRT-PCR assays for routine detection of SARS-CoV-2 have been published ,7. DetecN and NSP12 (RdRP1) genes used had no reported mutations. Thus, the selected primers/probes could detect all SARS-CoV-2 variants reported until today. In addition, targeting two viral genes, RdRP and N genes, would lower SARS-CoV-2 PCR false negative results owing to SARS-CoV mutations in the regions detected by SARS-CoV-2 PCR primers/probes sequences. Thus, selection of target genes is critical and the use of two or more gene targets in the diagnostic assays is essential to maximize diagnostic accuracy (sensitivity and specificity). These key points emphasize the importance of the appropriate PCR primers/probes sequences’ design strategy to best capture SARS-CoV-2 variants and the need for the diagnostic assay quality.Continuous mutations of the SARS-CoV-2 virus have been detected compared with Wuhan-Hu1 sequences or USA-WA1/2020, resulting in genetic variation in SARS-CoV-2 viral strains. This could potentially impact SARS-CoV-2 test performance. The FDA issued the Policy for Evaluating Impact of Viral Mutations on COVID-19 Tests . To asseTaq Polymerase antibody is used as the antibody is not available in large quantities and is expensive. To replace the Taq Polymerase antibody, an aptamer was tested to offer a cheaper and available alternative (5′-CAAGACGGGCGGGTGTGGTAGGCGCCCGTG-3′), as previously described [Taq Polymerase.The M-MLV RT (NCBI Reference Sequence: NP_057933.2) optimal temperature is 37 °C and thus would not provide a hot start capability for rRT-PCR. To overcome this limitation, the genetically modified clone for M-MLV RT was cloned and utilized in our in-house assay. The thermally stable Reverse Transcriptase was cloned with four mutations  that together increased the half-life of M-MLV at 50 °C and thus allowed hot start rRT-PCR. In addition, to provide a better hot start for the PCR reaction, an affordable alternative to the escribed . The aptTCEP is used in our in-house rRT-PCR master mix to replace DTT. DTT is a potent reducing agent widely used in molecular biology as an enzyme stabilizing agent by stabilizing proteins that possess free sulfhydryl groups ,16. ThusTaq polymerase resulted in an extremely reduced cost and increased the ability to manage the limitation of supplies should any pandemic arise in the future.Producing in-house rRT-PCR kits ensures a reduction in the expenses needed for the tests and makes them suitable for screening. Multiplexing also contributes to decreasing the expenses by testing multiple targets in one reaction in comparison with singleplex assays. Currently, there are many commercially available SARS-CoV-2 rRT-PCR kits available in the market. The prices range from $15 to $3 per test and the cost would increase substantially with shipping costs, on dry ice, taken into consideration. On the other hand, our in-house assay has an approximate cost of $0.2 per reaction. Nucleic acid extraction kits contribute to the cost of rRT-PCR; thus, an in-house RNA extraction procedure was established as well. Our in-house RNA extraction assay has an approximate cost of $0.5 per isolation. In conclusion, the in-house manufacturing of RNA isolation kits and rRT-PCR test with amplification enzymes, namely, thermostable reverse transcriptase, and In this study, ScriptTaq COVID PCR assay including kits for RNA isolation and multiplex rRT-PCR using in-house purified thermostable DNA polymerase and thermostable purified reverse transcriptase enzymes were established. Inclusivity of the primer/probe, RdRP gene, and N gene set used in the SARS-CoV-2 multiplex rRT-PCR assay was analyzed in silico and demonstrated 100% inclusivity for SARS-CoV-2 sequences and could detect all SARS-CoV-2 variants. Similarly, cross-reactivity of the SARS-CoV-2 multiplex rRT-PCR assay was evaluated by both in silico analysis and wet testing in clinical specimens. The in-silico mapping analysis of each primer/probe against 20 pathogens did not detect any cross-reactivity, while no cross reactivity was observed in clinical samples from patients with respiratory infections . The method had an overall agreement of 95.4%, a PPA of 90.2%, and an NPA of 100.0% with two authorized for emergency use only kits at two different tertiary hospitals in Riyadh. However, validation of the test using FDA-approved kits is needed and by other laboratories to assess the inter-laboratory test variation. The utilization of ScriptTaq COVID PCR assay would reduce the costs substantially and would help to combat the COVID-19 pandemic, especially in low-resource settings. The platform established in this study could be adopted for other viruses and diseases.


Deep water ports are human built coastal structures that by definition welcome ship traffic and disturbance. Evidence is accumulating that enhanced port activities such as dredging or deepening have negatively affected nearby natural habitats. Port Everglades Inlet (PEI) is a large active South Florida cargo port for over two million people and lies adjacent to coral reefs, dwindling mangroves, and recreational beaches. In this study, the microbial communities of PEI and adjacent reef sediments were characterized to serve as indicators for change due to dredging and assess anthropogenic influence on these sensitive ecosystems by sequencing the V4 region of 16S rRNA ahead of a large-scale port deepening event. For the first time, this study established baseline bacterial community characterizations and their patterns of diversity prior to and after a maintenance dredging event. PEI samples were collected for two consecutive years 2020  and 2021  from PEI sediments and adjacent coral reef sediments. In spite of their proximity and tidal connections through the PEI, reef and PEI sediment microbial communities were distinct. Changes in microbial diversity within the intracoastal waterway (ICW), a route for community exchange or transfers, were the greatest after maintenance dredging occurred. Microbial diversity in reef sediments also changed after dredging, indicating potential influence from resuspended sediments due to an associated increase in trace metals and decrease in cyanobacterial diversity. Sediments were identified as a possible source of human and coral pathogens, although dredging did not affect the relative abundances of these indicator microorganisms. This study highlighted the utility and relative ease of applying current molecular ecology methods to address macroscale questions with environmental management ramifications. PEI has been in operation for almost a century, harboring cargo ships in 1928 and federally authorized for passenger ships in 1930. Since then, this highly engineered deep water port is 1,923 feet (641 m) in length by 885 feet (295 m) wide with a depth of 39 feet . The PEI and adjacent intracoastal waterway (ICW) are subject to a high volume of commercial and recreational boat traffic and likely influenced by local and regional human-derived  anthropogenic inputs through a myriad of urban land uses well beyond its boundaries into its associated watershed. Within the next few years, the PEI will soon be one port looking to expand traffic by deepening and widening. This follows other developments across the globe to accommodate Neo-Panamax ships that were added to the fleet after the expansion of the Panama Canal in 2016 (https://www.usace.army). The Army Corp of Engineers released an Environmental Impact Statement (EIS) in late 2020 for public comment describing the possible effects of deep dredging from the current mean 42 feet (12.8 m) to a depth of 48 feet (14.6 m) in PEI (Port Everglades Inlet (PEI) is located on the East coast of Florida, U.S.A situated in three municipalities: Fort Lauderdale, Dania Beach, and Hollywood ( in 2016 . Port Ev) in PEI .Urban growth and port development can have detrimental effects on surrounding natural habitats, including coral reef systems . The S. Microbial communities provide important ecological and biogeochemical services, therefore microbial community profiling reveals valuable ecosystem information . When coIn this context, we aimed to characterize the microbial communities in the sediments of the PEI and the proximal coral reef before and after routine operational and maintenance (O&M) dredging events in January and March 2021. The baseline profiling was used to determine the resident microbiomes and microbial diversity of the PEI and reef sediments, and the changes that came after O&M dredging served as indicators of the potential influence of these activities. Microbiome information can be used by environmental managers to provide valuable context on the adverse effects that dredging on built environments with heavy anthropogenic influence may have on adjacent marine natural habitats .For this study, a total of 240 surface marine sediment samples from the top 10 cm were collected (120 per year in 2020 and 2021) from 40 sites spread over PEI (or port) and the adjacent Florida coral reef habitat to characterize the microbial community of sediment in these areas ; Fig. 1.All 240 sediment samples were processed for DNA analyses. Preparation of DNA samples from sediment and water acquired for high-throughput 16S rRNA gene sequencing were conducted as described in detail in previous studies . BrieflyMicrobial 16S rRNA gene libraries were generated by amplifying the V4 region with universal primers 515F and 806R with the Golay barcodes and Illumina adapters attached to the reverse primer following the protocol recommended by the Earth Microbiome Project . These pp value cut off of 0.05. Results were confirmed on square root transformed and untransformed data outside of CosmosID-HUB using function specpool as a part of the vegan package in R. The relative abundances of OTUs from CosmosID-HUB were used for beta diversity analyses, including SIMPER, ANOSIM, and non-metric multidimensional scaling (NMDS), which were performed using Primer-E v7  Sequence Read Archive (SRA). The NCBI SRA accession is PRJNA742832, release date: 2022-04-01.Metadata is available at our NSUworks repository: After QC and sequence processing, a total of 230 16S rRNA amplicon libraries were sequenced and analyzed from the PEI and the adjacent coral reef. The final 2020 dataset included 117 total samples (63 reef and 54 PEI samples), while the final 2021 dataset included 113 samples (51 PEI and 62 reef samples). All 230 samples had a sequence quality level of Q30 or above. Sequence results from 10 samples were omitted from further analyses when the read number fell below 50,000.p = 0.01) and the reef in 2021. Sites representing the channel  separated these groups. Reef sediments in 2020 clustered into two distinct groups that represented north (R01–R16) and south reef sites (R17–R22), which included control samples . This distinct clustering of the north and south reef sites was not observed in 2021  but there was a stronger difference between the reef and PEI sediments in 2021 . Indeed, differences between the south reef sediments in 2020 and 2021 were strong . The south reef sediments, including the control (R22), clustered with the north reef sediments in 2021 . These differences were due to the changes in microbial diversity in the ICW (sites P12–P18) from 2020 to 2021, which clustered distinctly from each other  and channel  from samples collected in the ICW. The control samples in the PEI, P01 and P18 where there was no dredging, still clustered with the PEI samples as they did in 2020.The differences between the PEI sediments in 2020 and 2021 were weak but significant . This corresponded to the decrease in the relative abundances of several families of Proteobacteria from 61.3 ± 5.4% to 50.4 ± 9.6%.The average similarity at the family level between PEI and reef sediments considering both years was 72.6% (SIMPER). Families from multiple phyla, including Desulfobacteraceae, Oxyphotobacteria, Desulfobulbaceae, Rhodospirillaceae, uncharacterized Gammaproteobacteria, Anaerolineaceae, and Thiotrichaceae, accounted for the top ~10% of the dissimilarities (SIMPER). Abundances of Anaerolineaceae, Thiotrichaceae, Desulfobacteraceae, and Desulfobulbaceae were higher in the PEI sediments and Oxyphotobacteria and Rhodospirillaceae were more abundant in the reef sediments. Cyanobacterial abundances decreased in the reef sediments in 2021, driving differences in microbial diversity between years at this location, especially in the south reef sites (SIMPER). Differences in the intracoastal in 2020 p = 0.10).Potential pathogens associated with Stony Coral Tissue Loss Disease (SCTLD) were identified, specifically from some of the most dominant orders observed in this study Rhizobiales and Rhodobacterales which were present in the sediments of both the PEI and reef before and after dredging  and S3. Staphylococcus, Streptococcus, Enterococcus aerogenes, and Vibrio. E. aerogenes  was ubiquitous in PEI and reef sediments. The results reflect recent surveys of waterborne bacterioplankton from same Broward County area . These include nty area .TN, TP, TC, and TOC and trace metals and elements were measured in a subset of samples to represent the chemical profiles of sediments in the PEI and reef  and S4B.2 = 0.533, p = 0.001). However, the bacterial assemblage of the sediments of the PEI and reef in 2020 alone correlated to Sb and TP , and in 2021, they strongly correlated to Be, Mo, and Pb . Pb also strongly and positively (R2 > 0.90) correlated to Hg, Zn, Cu, Ni, Co, and Al and therefore Pb was used as a proxy for this group of trace metals and minerals to correct for variation inflation.Several of these variables correlated to the differences in microbial communities . Se and https://floridadep.gov/rcp/coral/documents/analysis-sediments-port-everglades-inlet-pei-microbiome-characterization-phase) has provided an opportunity to characterize marine sediments from a heavily built (PEI) environment closely juxtaposed to a more natural (reef) and sensitive habitat.As one of five inlets in the three county  metropolis of S. Florida (U.S.A), the PEI, a commercial port, serves as a major connection between the ICW and the shallow coral reefs of S. Florida. PEI also lies adjacent to additional diverse habitat types–a depauperate mangrove preserve, urban and residential canals, and recreational beaches. This study  and the channel. Those changes in microbial community assemblages were associated with increased abundances of Chloroflexi and several phyla of Archaea and a decrease in Proteobacteria, which may be reflective of microbial community assemblages in the deeper marine sediments of the PEI. Indeed, Chloroflexi and many Archaea are obligate anaerobes and prevalent in anaerobic conditions that would be expected there. Their increase after dredging may have implications on biogeochemical cycles in the upper marine sediments in the PEI, as Archaea and Chloroflexi are important players in carbon, nitrogen and sulfur cycling  reported 2,779 wastewater spills in Florida in 2019, including a December 2019 burst in Fort Lauderdale that released 211.6 million gallons of raw sewage into canals and streets. Thus, close monitoring and determining the effects of urbanization, runoff, and subsequent water and sediment quality remain an ongoing concern for environmental protection managers and the wider public. There was no evidence that human pathogens increased in the PEI or reef sediments after maintenance dredging, however several were identified in the sediments at both locations. Again, this highlights the impact of human activities on natural ecosystems that ultimately extend to the coral reef habitat.Since 2014, SCTLD has been decimating coral populations on the Florida reef tract in Broward to the Florida Keys U.S.A, . PreviouCohaesibacter, Algicola, and Thalassobius. These taxa can also be found in sediments of the PEI and reef but were at relatively much lower abundances compared to Rhizobiales and Rhodobacterales. Although there was no evidence that these taxa increased after dredging, this points to the sediments as a possible source for coral pathogens in this area .his area . To advaf tracts . These dvs reef differences remain mostly consistent over 1 year and (iii) microbial beta diversity was more dynamic within each area, shifting potentially due to anthropogenic influence (dredging). There is evidence of possible disturbances via the O&M dredging within the PEI, primarily in the ICW, and possibly the reef sediments identified by distinct changes in microbial diversity and an increase in trace metal contamination. Although maintenance dredging did not increase the relative abundances of these taxa, evidence for the sediments being a potential source of both human and coral pathogens was also observed. This study highlighted the impact of human activity can drastically alter an environment and the possibility of impacts of surrounding natural environments. This is especially important to consider for environments that are sensitive to change, such as coral reefs and mangroves which are in very close proximity to many built and urban environments in South Florida, since their health is already degrading.This study using microbial diversity as an indicator to investigate the effects of dredging on a major port provides an interesting comparison of microbial diversity in a near century old human “built” environment (the PEI) adjacent to nearby “natural” and sensitive reef environment. The results from this study indicated that (i) microbial diversity between built and natural environments are distinct; (ii) these port 10.7717/peerj.14288/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.14288/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.14288/supp-3Supplemental Information 3The first three alphanumeric symbols of each ID match the sampling sites on the map of Figure 1.Click here for additional data file.10.7717/peerj.14288/supp-4Supplemental Information 4Symbol shapes and color match Figure 3. Clustering is denoted by the green solid lines at 80%.Click here for additional data file.10.7717/peerj.14288/supp-5Supplemental Information 5Samples are clustered based on abundance profiles.Click here for additional data file.10.7717/peerj.14288/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.14288/supp-7Supplemental Information 7The shading highlights the PEI samples taken for each element. If the shading is green, it indicates there were statistical differences in the concentration of that element in the PEI between 2020 and 2021, and the gray shading indicates there were no differences between 2020 and 2021 for that element in the PEI. An asterisk indicates if there were statistical differences in that element in the reef in after dredging in 2021. Statistically significant differences between the PEI and reef, or between years at either site were determined using one-way ANOVA with a p value cut-off of 0.05.Click here for additional data file.