AIM: To explore the new target genes transactivated by hepatitis C virus (HCV) core protein and to elucidate the pathogenesis of HCV infection. METHODS: Reverse transcribed cDNA was subjected to microarray assay. The ...AIM: To explore the new target genes transactivated by hepatitis C virus (HCV) core protein and to elucidate the pathogenesis of HCV infection. METHODS: Reverse transcribed cDNA was subjected to microarray assay. The coding gene transactivated by HCV core protein was cloned and analyzed with bioinformatics methods. RESULTS: The expressive vector of pcDNA3.1(-)-core was constructed and confirmed by restriction enzyme digestion and DNA sequencing and approved correct. mRNA was purified from HepGZ and HepG2 cells transfected with pcDNA3.1(-)-core, respectively. The cDNA derived was subjected to microarray assay. A new gene named HCTP4 was cloned with molecular biological method in combination with bioinformatics method. CONCLUSION: HCV core is a potential transactivator. Microarray is an efficient and convenient method for analysis of differentially expressed genes.展开更多
BACKGROUND The diagnosis of primary biliary cholangitis(PBC)remains challenging,particularly in cases where anti-mitochondrial antibody(AMA),anti-mitochondrial E2 subunit antibody(AMA-M2),anti-glycoprotein 210(anti-gp...BACKGROUND The diagnosis of primary biliary cholangitis(PBC)remains challenging,particularly in cases where anti-mitochondrial antibody(AMA),anti-mitochondrial E2 subunit antibody(AMA-M2),anti-glycoprotein 210(anti-gp210),and anti-speckled protein 100(anti-Sp100)are all negative.In such instances,the condition is often confirmed through a liver needle biopsy.AIM To identify additional plasma biomarkers for non-invasive diagnostic methods of PBC.METHODS We utilized the Sengenics KREX^(TM)immunome protein array to identify potential biomarkers for the diagnosis of PBC.Subsequently,we validated the predictive capability of the RPL30 antibody through an ELISA and retrospectively analyzed its association with the clinical features of 17 autoantibody-negative PBC cases and 45 autoantibody-positive PBC cases.RESULTS In our study we observed that RPL30 demonstrated the highest fold-change difference in PBC,with a penetrance frequency of 40%and a penetrance fold change of 38.30147.The analysis of anti-RPL30 optical density values between patients with AMA/AMA-M2/anti-gp210/anti-Sp100-negative PBC(autoantibody-negative PBC)and healthy controls using a receiver operating characteristic curve yielded an area under the curve of 0.853.This analysis established an optimal cutoff value of 0.0708,achieving 100%specificity and 75%sensitivity.The combination of anti-RPL30 and other autoantibodies elevated the diagnosis rate of PBC from 61.29%to 79.00%(P=0.0489).Anti-RPL30 demonstrated a high positive rate in antibody-negative PBC cases,including AMA/AMAM2/anti-gp210/anti-Sp100-negative cases.Correlation analysis of anti-RPL30 optical density values with clinical data from patients with PBC revealed a positive association with both the international normalized ratio(P=0.008)and the Model for End-Stage Liver Disease score(P=0.003).CONCLUSION Our study highlighted the potential of anti-RPL30 as a promising biomarker for diagnosing PBC,particularly in autoantibody-negative cases.展开更多
Objective:In the realm of Class I pathogens,Burkholderia pseudomallei(BP)stands out for its propensity to induce severe pathogenicity.Investigating the intricate interactions between BP and host cells is imperative fo...Objective:In the realm of Class I pathogens,Burkholderia pseudomallei(BP)stands out for its propensity to induce severe pathogenicity.Investigating the intricate interactions between BP and host cells is imperative for comprehending the dynamics of BP infection and discerning biomarkers indicative of the host cell response process.Methods:mRNA extraction from BP-infected mouse macrophages constituted the initial step of our study.Employing gene expression arrays,the extracted RNA underwent conversion into digital signals.The percentile shift method facilitated data processing,with the identification of genes manifesting significant differences accomplished through the application of the t-test.Subsequently,a comprehensive analysis involving Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway was conducted on the differentially expressed genes(DEGs).Leveraging the ESTIMATE algorithm,gene signatures were utilized to compute risk scores for gene expression data.Support vector machine analysis and gene enrichment scores were instrumental in establishing correlations between biomarkers and macrophages,followed by an evaluation of the predictive power of the identified biomarkers.Results:The functional and pathway associations of the DEGs predominantly centered around G protein-coupled receptors.A noteworthy positive correlation emerged between the blue module,consisting of 416 genes,and the StromaScore.FZD4,identified through support vector machine analysis among intersecting genes,indicated a robust interaction with macrophages,suggesting its potential as a robust biomarker.FZD4 exhibited commendable predictive efficacy,with BP infection inducing its expression in both macrophages and mouse lung tissue.Western blotting in macrophages confirmed a significant upregulation of FZD4 expression from 0.5 to 24 h post-infection.In mouse lung tissue,FZD4 manifested higher expression in the cytoplasm of pulmonary epithelial cells in BP-infected lungs than in the control group.Conclusion:Thesefindings underscore the upregulation of FZD4 expression by BP in both macrophages and lung tissue,pointing to its prospective role as a biomarker in the pathogenesis of BP infection.展开更多
While the peripheral nervous system has regenerative ability,restoration of sufficient function remains a challenge.Vimentin has been shown to be localized in axonal growth fronts and associated with nerve regeneratio...While the peripheral nervous system has regenerative ability,restoration of sufficient function remains a challenge.Vimentin has been shown to be localized in axonal growth fronts and associated with nerve regeneration,including myelination,neuroplasticity,kinase signaling in nerve axoplasm,and cell migration;however,the mechanisms regulating its expression within Schwann cell（SC） remain unexplored.The aim of this study was to profile the spatial and temporal expression profile of micro RNA（mi RNA） in a regenerating rat sciatic nerve after transection,and explore the potential role of mi R-138-5 p targeting vimentin in SC proliferation and migration.A rat sciatic nerve transection model,utilizing a polyethylene nerve guide,was used to investigate mi RNA expression at 7,14,30,60,and 90 days during nerve regeneration.Relative levels of mi RNA expression were determined using microarray analysis and subsequently validated with quantitative real-time polymerase chain reaction.In vitro assays were conducted with cultured Schwann cells transfected with mi RNA mimics and assessed for migratory and proliferative potential.The top seven dysregulated mi RNAs reported in this study have been implicated in cell migration elsewhere,and GO and KEGG analyses predicted activities essential to wound healing.Transfection of one of these,mi RNA-138-5 p,into SCs reduced cell migration and proliferation.mi R-138-5 p has been shown to directly target vimentin in cancer cells,and the luciferase assay performed here in rat Schwann cells confirmed it.These results detail a role of mi R-138-5 p in rat peripheral nerve regeneration and expand on reports of it as an important regulator in the peripheral nervous system.展开更多
Saline-alkali soil seriously threatens agriculture productivity; therefore, understanding the mechanism of plant tolerance to alkaline-salt stress has become a major challenge. Halophytic Puccinellia tenuiflora can to...Saline-alkali soil seriously threatens agriculture productivity; therefore, understanding the mechanism of plant tolerance to alkaline-salt stress has become a major challenge. Halophytic Puccinellia tenuiflora can tolerate salt and alkaline-salt stress, and is thus an ideal plant for studying this tolerance mechanism. In this study, we examined the salt and alkaline-salt stress tolerance of P. tenuiflora, and analyzed gene expression profiles under these stresses. Physiological experiments revealed that P. tenuiflora can grow normally with maximum stress under 600 mmol/L NaCl and 150 mmol/L Na 2 CO 3 （pH 11.0） for 6 d. We identified 4,982 unigenes closely homologous to rice and barley. Furthermore, 1,105 genes showed differentially expressed profiles under salt and alkaline-salt treatments. Differentially expressed genes were overrepresented in functions of photosynthesis, oxidation reduction, signal transduction, and transcription regulation. Almost all genes downregulated under salt and alkaline-salt stress were related to cell structure, photosynthesis, and protein synthesis. Comparing with salt stress, alkaline-salt stress triggered more differentially expressed genes and significantly upregulated genes related to H ＋ transport and citric acid synthesis. These data indicate common and diverse features of salt and alkaline-salt stress tolerance, and give novel insights into the molecular and physiological mechanisms of plant salt and alkaline-salt tolerance.展开更多
Background: Autophagy of alveolar macrophages is a crucial process in ischemia/reperfusion injury-induced acute lung injury (ALI). Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent cells with the po...Background: Autophagy of alveolar macrophages is a crucial process in ischemia/reperfusion injury-induced acute lung injury (ALI). Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent cells with the potential for repairing injured sites and regulating autophagy. This study was to investigate the influence of BM-MSCs on autophagy of macrophages in the oxygen-glucose deprivation/restoration (OGD/R) microenvironment and to explore the potential mechanism.Methods: We established a co-culture system of macrophages (RAW264.7) with BM-MSCs under OGD/R conditionsin vitro. RAW264.7 cells were transfected with recombinant adenovirus (Ad-mCherry-GFP-LC3B) and autophagic status of RAW264.7 cells was observed under a fluorescence microscope. Autophagy-related proteins light chain 3 (LC3)-I, LC3-II, and p62 in RAW264.7 cells were detected by Western blotting. We used microarray expression analysis to identify the differently expressed genes between OGD/R treated macrophages and macrophages co-culture with BM-MSCs. We investigated the gene heme oxygenase-1 (HO-1), which is downstream of the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway.Results: The ratio of LC3-II/LC3-I of OGD/R treated RAW264.7 cells was increased (1.27 ± 0.20vs. 0.44 ± 0.08,t = 6.67,P < 0.05), while the expression of p62 was decreased (0.77 ± 0.04vs. 0.95 ± 0.10,t = 2.90,P < 0.05), and PI3K (0.40 ± 0.06vs. 0.63 ± 0.10,t = 3.42,P < 0.05) and p-Akt/Akt ratio was also decreased (0.39 ± 0.02vs. 0.58 ± 0.03,t = 9.13,P < 0.05). BM-MSCs reduced the LC3-II/LC3-I ratio of OGD/R treated RAW264.7 cells (0.68 ± 0.14vs. 1.27 ± 0.20,t = 4.12,P < 0.05), up-regulated p62 expression (1.10 ± 0.20vs. 0.77 ± 0.04,t = 2.80,P < 0.05), and up-regulated PI3K (0.54 ± 0.05vs. 0.40 ± 0.06,t = 3.11,P < 0.05) and p-Akt/Akt ratios (0.52 ± 0.05vs. 0.39 ± 0.02,t = 9.13,P < 0.05). A whole-genome microarray assay screened the differentially expressed geneHO-1, which is downstream of the PI3K/Akt signaling pathway, and the alteration ofHO-1 mRNA and protein expression was consistent with the data on PI3K/Akt pathway.Conclusions: Our results suggest the existence of the PI3K/Akt/HO-1 signaling pathway in RAW264.7 cells under OGD/R circumstancesin vitro, revealing the mechanism underlying BM-MSC-mediated regulation of autophagy and enriching the understanding of potential therapeutic targets for the treatment of ALI.展开更多
We aimed to develop a whole-genome sequencing(WGS)-based copy number variant(CNV)calling algorithm with the potential of replacing chromosomal microarray assay(CMA)for clinical diagnosis.JAX-CNV is thus developed for ...We aimed to develop a whole-genome sequencing(WGS)-based copy number variant(CNV)calling algorithm with the potential of replacing chromosomal microarray assay(CMA)for clinical diagnosis.JAX-CNV is thus developed for CNV detection from WGS data.The performance of this CNV calling algorithm was evaluated in a blinded manner on 31 samples and compared to the 112 CNVs reported by clinically validated CMAs for these 31 samples.The result showed that JAX-CNV recalled 100%of these CNVs.Besides,JAX-CNV identified an average of 30 CNVs per individual,representing an approximately seven-fold increase compared to calls of clinically validated CMAs.Experimental validation of 24 randomly selected CNVs showed one false positive,i.e.,a false discovery rate(FDR)of 4.17%.A robustness test on lowercoverage data revealed a 100%sensitivity for CNVs larger than 300 kb(the current threshold for College of American Pathologists)down to 10×coverage.For CNVs larger than 50 kb,sensitivities were 100%for coverages deeper than 20×,97%for 15×,and 95%for 10×.We developed a WGS-based CNV pipeline,including this newly developed CNV caller JAX-CNV,and found it capable of detecting CMA-reported CNVs at a sensitivity of 100%with about a FDR of 4%.We propose that JAX-CNV could be further examined in a multi-institutional study to justify the transition of first-tier genetic testing from CMAs to WGS.JAX-CNV is available at https://github.com/TheJacksonLaboratory/JAX-CNV.展开更多
基金Supported by the National Natural Science Foundation of China, No. 39970674
文摘AIM: To explore the new target genes transactivated by hepatitis C virus (HCV) core protein and to elucidate the pathogenesis of HCV infection. METHODS: Reverse transcribed cDNA was subjected to microarray assay. The coding gene transactivated by HCV core protein was cloned and analyzed with bioinformatics methods. RESULTS: The expressive vector of pcDNA3.1(-)-core was constructed and confirmed by restriction enzyme digestion and DNA sequencing and approved correct. mRNA was purified from HepGZ and HepG2 cells transfected with pcDNA3.1(-)-core, respectively. The cDNA derived was subjected to microarray assay. A new gene named HCTP4 was cloned with molecular biological method in combination with bioinformatics method. CONCLUSION: HCV core is a potential transactivator. Microarray is an efficient and convenient method for analysis of differentially expressed genes.
基金Supported by National Natural Science Foundation of China,No.82172257Health Care Major Project of Guangzhou,No.202206080001Science and Technology Cooperation Program of Fujian Province,No.2021I0036。
文摘BACKGROUND The diagnosis of primary biliary cholangitis(PBC)remains challenging,particularly in cases where anti-mitochondrial antibody(AMA),anti-mitochondrial E2 subunit antibody(AMA-M2),anti-glycoprotein 210(anti-gp210),and anti-speckled protein 100(anti-Sp100)are all negative.In such instances,the condition is often confirmed through a liver needle biopsy.AIM To identify additional plasma biomarkers for non-invasive diagnostic methods of PBC.METHODS We utilized the Sengenics KREX^(TM)immunome protein array to identify potential biomarkers for the diagnosis of PBC.Subsequently,we validated the predictive capability of the RPL30 antibody through an ELISA and retrospectively analyzed its association with the clinical features of 17 autoantibody-negative PBC cases and 45 autoantibody-positive PBC cases.RESULTS In our study we observed that RPL30 demonstrated the highest fold-change difference in PBC,with a penetrance frequency of 40%and a penetrance fold change of 38.30147.The analysis of anti-RPL30 optical density values between patients with AMA/AMA-M2/anti-gp210/anti-Sp100-negative PBC(autoantibody-negative PBC)and healthy controls using a receiver operating characteristic curve yielded an area under the curve of 0.853.This analysis established an optimal cutoff value of 0.0708,achieving 100%specificity and 75%sensitivity.The combination of anti-RPL30 and other autoantibodies elevated the diagnosis rate of PBC from 61.29%to 79.00%(P=0.0489).Anti-RPL30 demonstrated a high positive rate in antibody-negative PBC cases,including AMA/AMAM2/anti-gp210/anti-Sp100-negative cases.Correlation analysis of anti-RPL30 optical density values with clinical data from patients with PBC revealed a positive association with both the international normalized ratio(P=0.008)and the Model for End-Stage Liver Disease score(P=0.003).CONCLUSION Our study highlighted the potential of anti-RPL30 as a promising biomarker for diagnosing PBC,particularly in autoantibody-negative cases.
基金The study was supported by Yuying Program Incubation Project of General Hospital of Center Theater(ZZYFH202104)Wuhan Young and Middle-Aged Medical Backbone Talent Project 2020(2020-55)Logistics Research Program Project 2019(CLB19J029).
文摘Objective:In the realm of Class I pathogens,Burkholderia pseudomallei(BP)stands out for its propensity to induce severe pathogenicity.Investigating the intricate interactions between BP and host cells is imperative for comprehending the dynamics of BP infection and discerning biomarkers indicative of the host cell response process.Methods:mRNA extraction from BP-infected mouse macrophages constituted the initial step of our study.Employing gene expression arrays,the extracted RNA underwent conversion into digital signals.The percentile shift method facilitated data processing,with the identification of genes manifesting significant differences accomplished through the application of the t-test.Subsequently,a comprehensive analysis involving Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway was conducted on the differentially expressed genes(DEGs).Leveraging the ESTIMATE algorithm,gene signatures were utilized to compute risk scores for gene expression data.Support vector machine analysis and gene enrichment scores were instrumental in establishing correlations between biomarkers and macrophages,followed by an evaluation of the predictive power of the identified biomarkers.Results:The functional and pathway associations of the DEGs predominantly centered around G protein-coupled receptors.A noteworthy positive correlation emerged between the blue module,consisting of 416 genes,and the StromaScore.FZD4,identified through support vector machine analysis among intersecting genes,indicated a robust interaction with macrophages,suggesting its potential as a robust biomarker.FZD4 exhibited commendable predictive efficacy,with BP infection inducing its expression in both macrophages and mouse lung tissue.Western blotting in macrophages confirmed a significant upregulation of FZD4 expression from 0.5 to 24 h post-infection.In mouse lung tissue,FZD4 manifested higher expression in the cytoplasm of pulmonary epithelial cells in BP-infected lungs than in the control group.Conclusion:Thesefindings underscore the upregulation of FZD4 expression by BP in both macrophages and lung tissue,pointing to its prospective role as a biomarker in the pathogenesis of BP infection.
文摘While the peripheral nervous system has regenerative ability,restoration of sufficient function remains a challenge.Vimentin has been shown to be localized in axonal growth fronts and associated with nerve regeneration,including myelination,neuroplasticity,kinase signaling in nerve axoplasm,and cell migration;however,the mechanisms regulating its expression within Schwann cell（SC） remain unexplored.The aim of this study was to profile the spatial and temporal expression profile of micro RNA（mi RNA） in a regenerating rat sciatic nerve after transection,and explore the potential role of mi R-138-5 p targeting vimentin in SC proliferation and migration.A rat sciatic nerve transection model,utilizing a polyethylene nerve guide,was used to investigate mi RNA expression at 7,14,30,60,and 90 days during nerve regeneration.Relative levels of mi RNA expression were determined using microarray analysis and subsequently validated with quantitative real-time polymerase chain reaction.In vitro assays were conducted with cultured Schwann cells transfected with mi RNA mimics and assessed for migratory and proliferative potential.The top seven dysregulated mi RNAs reported in this study have been implicated in cell migration elsewhere,and GO and KEGG analyses predicted activities essential to wound healing.Transfection of one of these,mi RNA-138-5 p,into SCs reduced cell migration and proliferation.mi R-138-5 p has been shown to directly target vimentin in cancer cells,and the luciferase assay performed here in rat Schwann cells confirmed it.These results detail a role of mi R-138-5 p in rat peripheral nerve regeneration and expand on reports of it as an important regulator in the peripheral nervous system.
基金supported by a grant from the Chinese Academy of Sciences (No. KSCX3-EW-N-07-3)
文摘Saline-alkali soil seriously threatens agriculture productivity; therefore, understanding the mechanism of plant tolerance to alkaline-salt stress has become a major challenge. Halophytic Puccinellia tenuiflora can tolerate salt and alkaline-salt stress, and is thus an ideal plant for studying this tolerance mechanism. In this study, we examined the salt and alkaline-salt stress tolerance of P. tenuiflora, and analyzed gene expression profiles under these stresses. Physiological experiments revealed that P. tenuiflora can grow normally with maximum stress under 600 mmol/L NaCl and 150 mmol/L Na 2 CO 3 （pH 11.0） for 6 d. We identified 4,982 unigenes closely homologous to rice and barley. Furthermore, 1,105 genes showed differentially expressed profiles under salt and alkaline-salt treatments. Differentially expressed genes were overrepresented in functions of photosynthesis, oxidation reduction, signal transduction, and transcription regulation. Almost all genes downregulated under salt and alkaline-salt stress were related to cell structure, photosynthesis, and protein synthesis. Comparing with salt stress, alkaline-salt stress triggered more differentially expressed genes and significantly upregulated genes related to H ＋ transport and citric acid synthesis. These data indicate common and diverse features of salt and alkaline-salt stress tolerance, and give novel insights into the molecular and physiological mechanisms of plant salt and alkaline-salt tolerance.
基金National Natural Science Foundation of China(No.81490533)。
文摘Background: Autophagy of alveolar macrophages is a crucial process in ischemia/reperfusion injury-induced acute lung injury (ALI). Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent cells with the potential for repairing injured sites and regulating autophagy. This study was to investigate the influence of BM-MSCs on autophagy of macrophages in the oxygen-glucose deprivation/restoration (OGD/R) microenvironment and to explore the potential mechanism.Methods: We established a co-culture system of macrophages (RAW264.7) with BM-MSCs under OGD/R conditionsin vitro. RAW264.7 cells were transfected with recombinant adenovirus (Ad-mCherry-GFP-LC3B) and autophagic status of RAW264.7 cells was observed under a fluorescence microscope. Autophagy-related proteins light chain 3 (LC3)-I, LC3-II, and p62 in RAW264.7 cells were detected by Western blotting. We used microarray expression analysis to identify the differently expressed genes between OGD/R treated macrophages and macrophages co-culture with BM-MSCs. We investigated the gene heme oxygenase-1 (HO-1), which is downstream of the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway.Results: The ratio of LC3-II/LC3-I of OGD/R treated RAW264.7 cells was increased (1.27 ± 0.20vs. 0.44 ± 0.08,t = 6.67,P < 0.05), while the expression of p62 was decreased (0.77 ± 0.04vs. 0.95 ± 0.10,t = 2.90,P < 0.05), and PI3K (0.40 ± 0.06vs. 0.63 ± 0.10,t = 3.42,P < 0.05) and p-Akt/Akt ratio was also decreased (0.39 ± 0.02vs. 0.58 ± 0.03,t = 9.13,P < 0.05). BM-MSCs reduced the LC3-II/LC3-I ratio of OGD/R treated RAW264.7 cells (0.68 ± 0.14vs. 1.27 ± 0.20,t = 4.12,P < 0.05), up-regulated p62 expression (1.10 ± 0.20vs. 0.77 ± 0.04,t = 2.80,P < 0.05), and up-regulated PI3K (0.54 ± 0.05vs. 0.40 ± 0.06,t = 3.11,P < 0.05) and p-Akt/Akt ratios (0.52 ± 0.05vs. 0.39 ± 0.02,t = 9.13,P < 0.05). A whole-genome microarray assay screened the differentially expressed geneHO-1, which is downstream of the PI3K/Akt signaling pathway, and the alteration ofHO-1 mRNA and protein expression was consistent with the data on PI3K/Akt pathway.Conclusions: Our results suggest the existence of the PI3K/Akt/HO-1 signaling pathway in RAW264.7 cells under OGD/R circumstancesin vitro, revealing the mechanism underlying BM-MSC-mediated regulation of autophagy and enriching the understanding of potential therapeutic targets for the treatment of ALI.
基金supported in part by the operational funds from The First Affiliated Hospital of Xi’an Jiaotong University, Chinasupported by the National Institutes of Health, USA (Grant Nos. U24AG041689 and U54AG052427)+5 种基金supported by the National Natural Science Foundation of China (Grant Nos. 61702406 and 31671372)the National Science and Technology Major Project of China (Grant No. 2018ZX10302205)the National Key R&D Program of China (Grant Nos. 2018YFC0910400 and 2017YFC0907500)the General Financial Grant from the China Postdoctoral Science Foundation (Grant No. 2017M623178)supported in part by the Ewha Womans University Research, South Korea (Grant No. 2018-2019)supported in part by the Connecticut Bio-Innovative Fund, USA
文摘We aimed to develop a whole-genome sequencing(WGS)-based copy number variant(CNV)calling algorithm with the potential of replacing chromosomal microarray assay(CMA)for clinical diagnosis.JAX-CNV is thus developed for CNV detection from WGS data.The performance of this CNV calling algorithm was evaluated in a blinded manner on 31 samples and compared to the 112 CNVs reported by clinically validated CMAs for these 31 samples.The result showed that JAX-CNV recalled 100%of these CNVs.Besides,JAX-CNV identified an average of 30 CNVs per individual,representing an approximately seven-fold increase compared to calls of clinically validated CMAs.Experimental validation of 24 randomly selected CNVs showed one false positive,i.e.,a false discovery rate(FDR)of 4.17%.A robustness test on lowercoverage data revealed a 100%sensitivity for CNVs larger than 300 kb(the current threshold for College of American Pathologists)down to 10×coverage.For CNVs larger than 50 kb,sensitivities were 100%for coverages deeper than 20×,97%for 15×,and 95%for 10×.We developed a WGS-based CNV pipeline,including this newly developed CNV caller JAX-CNV,and found it capable of detecting CMA-reported CNVs at a sensitivity of 100%with about a FDR of 4%.We propose that JAX-CNV could be further examined in a multi-institutional study to justify the transition of first-tier genetic testing from CMAs to WGS.JAX-CNV is available at https://github.com/TheJacksonLaboratory/JAX-CNV.
