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Epigenomic annotations for the rat lag far behind those of human and mouse, despite the rat’s immense utility in pharmacological and behavioral studies and the need to understand their epigenetic mechanisms. We have designed a targeted-enrichment method followed by next-generation sequencing (Methyl-Seq) to identify DNA methylation (DNAm) signatures across the rat genome. The design reflected an attempt to create a more comprehensive investigation of the rat epigenome, as it included promoters, CpG islands, and island shores of all RefSeq genes. In this study, we implemented the rat Methyl-Seq platform and tested its ability to distinguish differentially methylated regions (DMRs) among three different tissue types, three distinct brain regions, and, in the hippocampus, between males and females. These comparisons yielded DNAm differences of differing magnitudes, many of which were independently validated by bisulfite pyrosequencing, including autosomal regions that were predicted to show the least degree of difference in DNAm between males and females. Quantitative reverse transcription PCR revealed that most genes associated with the DMRs showed tissue-, brain region-, and sex-specific differences in expression. In particular, we found evidence for sex-specific DNAm and expression differences at Tubb6, Lrrn2, Tex26, and Sox5l1, all of which play important roles in neurodevelopment and have been implicated in studies examining sex differences. Our results demonstrate the utility of the rat Methyl-Seq platform and suggest the presence of DNAm differences between the male and female hippocampus. The rat Methyl-Seq has the potential to provide epigenomic insights into pharmacological and behavioral studies performed in the rat.

Abstract Background Reproducible detection of inherited variants with whole genome sequencing (WGS) is vital for the implementation of precision medicine and is a complicated process in which each step affects variant call quality. Systematically assessing reproducibility of inherited variants with WGS and impact of each step in the process is needed for understanding and improving quality of inherited variants from WGS. Results To dissect the impact of factors involved in detection of inherited variants with WGS, we sequence triplicates of eight DNA samples representing two populations on three short-read sequencing platforms using three library kits in six labs and call variants with 56 combinations of aligners and callers. We find that bioinformatics pipelines (callers and aligners) have a larger impact on variant reproducibility than WGS platform or library preparation. Single-nucleotide variants (SNVs), particularly outside difficult-to-map regions, are more reproducible than small insertions and deletions (indels), which are least reproducible when > 5 bp. Increasing sequencing coverage improves indel reproducibility but has limited impact on SNVs above 30×. Conclusions Our findings highlight sources of variability in variant detection and the need for improvement of bioinformatics pipelines in the era of precision medicine with WGS.

Abstract Background Oncopanel genomic testing, which identifies important somatic variants, is increasingly common in medical practice and especially in clinical trials. Currently, there is a paucity of reliable genomic reference samples having a suitably large number of pre-identified variants for properly assessing oncopanel assay analytical quality and performance. The FDA-led Sequencing and Quality Control Phase 2 (SEQC2) consortium analyze ten diverse cancer cell lines individually and their pool, termed Sample A, to develop a reference sample with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance. Results In reference Sample A, we identify more than 40,000 variants down to 1% allele frequency with more than 25,000 variants having less than 20% allele frequency with 1653 variants in COSMIC-related genes. This is 5–100× more than existing commercially available samples. We also identify an unprecedented number of negative positions in coding regions, allowing statistical rigor in assessing limit-of-detection, sensitivity, and precision. Over 300 loci are randomly selected and independently verified via droplet digital PCR with 100% concordance. Agilent normal reference Sample B can be admixed with Sample A to create new samples with a similar number of known variants at much lower allele frequency than what exists in Sample A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels. Conclusion These new reference samples and their admixtures provide superior capability for performing oncopanel quality control, analytical accuracy, and validation for small to large oncopanels and liquid biopsy assays.

Abstract Background Targeted sequencing using oncopanels requires comprehensive assessments of accuracy and detection sensitivity to ensure analytical validity. By employing reference materials characterized by the U.S. Food and Drug Administration-led SEquence Quality Control project phase2 (SEQC2) effort, we perform a cross-platform multi-lab evaluation of eight Pan-Cancer panels to assess best practices for oncopanel sequencing. Results All panels demonstrate high sensitivity across targeted high-confidence coding regions and variant types for the variants previously verified to have variant allele frequency (VAF) in the 5–20% range. Sensitivity is reduced by utilizing VAF thresholds due to inherent variability in VAF measurements. Enforcing a VAF threshold for reporting has a positive impact on reducing false positive calls. Importantly, the false positive rate is found to be significantly higher outside the high-confidence coding regions, resulting in lower reproducibility. Thus, region restriction and VAF thresholds lead to low relative technical variability in estimating promising biomarkers and tumor mutational burden. Conclusion This comprehensive study provides actionable guidelines for oncopanel sequencing and clear evidence that supports a simplified approach to assess the analytical performance of oncopanels. It will facilitate the rapid implementation, validation, and quality control of oncopanels in clinical use.

Abstract Olfactomedin 4 (OLFM4) is expressed in normal prostate epithelial cells and immortalized normal human prostate epithelial cells (RWPE1), but the identity of OLFM4-expressing cells within these populations and OLFM4’s physiological functions in these cells have not been elucidated. Using single-cell RNA sequencing analysis, we found here that OLFM4 was expressed in multiple stem/progenitor-like cell populations in both the normal prostate epithelium and RWPE1 cells and was frequently co-expressed with KRT13 and LY6D in RWPE1 cells. Functionally, OLFM4-knockout RWPE1 cells exhibited enhanced proliferation of the stem/progenitor-like cell population, shifts stem/progenitor-like cell division to favor symmetric division and differentiated into higher levels PSA expression cells in organoid assays compared with OLFM4-wild RWPE1 cells. Bulk-cell RNA sequencing analysis pinpointed that cMYC expression were enhanced in the OLFM4-knockout RWPE1 cells compared with OLFM4-wild cells. Molecular and signaling pathway studies revealed an increase in the WNT/APC/MYC signaling pathway gene signature, as well as that of MYC target genes that regulate multiple biological processes, in OLFM4-knockout RWPE1 cells. These findings indicated that OLFM4 is co-expressed with multiple stem/progenitor cell marker genes in prostate epithelial cells and acts as a novel mediator in prostate stem/progenitor cell proliferation and differentiation.

Majority of human long non-coding RNAs (lncRNAs) are not conserved in mouse. Here the authors identify metabolic trait-associated lncRNA genes and show a functional role of a non-conserved human lncRNA, LINC01018, in lipid metabolism using a humanized mouse model.

BACKGROUND Fasting and NAD+-boosting compounds, including NAD+ precursor nicotinamide riboside (NR), confer antiinflammatory effects. However, the underlying mechanisms and therapeutic potential are incompletely defined.METHODS We explored the underlying biology in myeloid cells from healthy volunteers following in vivo placebo or NR administration and subsequently tested the findings in vitro in monocytes extracted from patients with systemic lupus erythematosus (SLE).RESULTS RNA-Seq of unstimulated and LPS-activated monocytes implicated NR in the regulation of autophagy and type I IFN signaling. In primary monocytes, NR blunted LPS-induced IFN-β production, and genetic or pharmacological disruption of autophagy phenocopied this effect. Given that NAD+ is a coenzyme in oxidoreductive reactions, metabolomics was performed and identified that NR increased the inosine level. Inosine supplementation similarly blunted autophagy and IFN-β release. Finally, because SLE exhibits type I IFN dysregulation, we assessed the NR effect on monocytes from patients with SLE and found that NR reduced autophagy and IFN-β release.CONCLUSION We conclude that NR, in an NAD+-dependent manner and in part via inosine signaling, mediated suppression of autophagy and attenuated type I IFN in myeloid cells, and we identified NR as a potential adjunct for SLE management.TRIAL REGISTRATION ClinicalTrials.gov registration numbers NCT02812238, NCT00001846, and NCT00001372.FUNDING This work was supported by the NHLBI and NIAMS Intramural Research divisions.

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a widely available curative option for patients with sickle cell disease (SCD). Our original non-myeloablative haplo-HSCT trial employing post-transplant (PT) cyclophosphamide had a low incidence of GVHD but had high rejection rates. Here, we aimed to evaluate immune reconstitution following haplo-HSCT and identify cytokines and cells associated with graft rejection/engraftment. 50 cytokines and 10 immune cell subsets were screened using multiplex-ELISA and flow cytometry, respectively, at baseline and PT-Days 30, 60, 100, and 180. We observed the most significant differences in cytokine levels between the engrafted and rejected groups at PT-Day 60, corresponding with clinical findings of secondary graft rejection. Of the 44 cytokines evaluated, plasma concentrations of 19 cytokines were different between the two groups at PT-Day 60. Factor analysis suggested two independent factors. The first factor (IL-17A, IL-10, IL-7, G-CSF, IL-2, MIP-1a, VEGF, and TGFb1 contributed significantly) was strongly associated with engraftment with OR = 2.7 (95%CI of 1.4 to 5.4), whereas the second factor (GROa and IL-18 contributed significantly) was not significantly associated with engraftment. Sufficient donor myeloid chimerism (DMC) is critical for the success of HSCT; here, we evaluated immune cells among high (H) DMC (DMC≥20%) and low (L) DMC (DMC

Summary: Psoriasis is an inflammatory skin disease associated with increased cardiovascular risk and serves as a reliable model to study inflammatory atherogenesis. Because neutrophils are implicated in atherosclerosis development, this study reports that the interaction among low-density granulocytes, a subset of neutrophils, and platelets is associated with noncalcified coronary plaque burden assessed by coronary computed tomography angiography. Because early atherosclerotic noncalcified burden can lead to fatal myocardial infarction, the low-density granulocyte−platelet interaction may play a crucial target for clinical intervention. Key Words: cardiovascular disease, low-density granulocytes, neutrophils, platelets, psoriasis

INTRODUCTION The clinical course of coronavirus 2019 (COVID-19) is heterogeneous, ranging from mild to severe multiorgan failure and death. In this study, we analyzed cell-free DNA (cfDNA) as a biomarker of injury to define the sources of tissue injury that contribute to such different trajectories.METHODS We conducted a multicenter prospective cohort study to enroll patients with COVID-19 and collect plasma samples. Plasma cfDNA was subject to bisulfite sequencing. A library of tissue-specific DNA methylation signatures was used to analyze sequence reads to quantitate cfDNA from different tissue types. We then determined the correlation of tissue-specific cfDNA measures to COVID-19 outcomes. Similar analyses were performed for healthy controls and a comparator group of patients with respiratory syncytial virus and influenza.RESULTS We found markedly elevated levels and divergent tissue sources of cfDNA in COVID-19 patients compared with patients who had influenza and/or respiratory syncytial virus and with healthy controls. The major sources of cfDNA in COVID-19 were hematopoietic cells, vascular endothelium, hepatocytes, adipocytes, kidney, heart, and lung. cfDNA levels positively correlated with COVID-19 disease severity, C-reactive protein, and D-dimer. cfDNA profile at admission identified patients who subsequently required intensive care or died during hospitalization. Furthermore, the increased cfDNA in COVID-19 patients generated excessive mitochondrial ROS (mtROS) in renal tubular cells in a concentration-dependent manner. This mtROS production was inhibited by a TLR9-specific antagonist.CONCLUSION cfDNA maps tissue injury that predicts COVID-19 outcomes and may mechanistically propagate COVID-19–induced tissue injury.FUNDING Intramural Targeted Anti–COVID-19 grant, NIH.

Analyzing host cells' transcriptional response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection will help delineate biological processes underlying viral pathogenesis. First, analysis of expression profiles of lung cell lines A549 and Calu3 revealed upregulation of antiviral interferon signaling genes in response to all three SARS-CoV-2, MERS-CoV, or influenza A virus (IAV) infections. However, perturbations in expression of genes involved in inflammatory, mitochondrial, and autophagy processes were specifically observed in SARS-CoV-2-infected cells. Next, a validation study in infected human nasopharyngeal samples also revealed perturbations in autophagy and mitochondrial processes. Specifically, mTOR expression, mitochondrial ribosomal, mitochondrial complex I, lysosome acidification, and mitochondrial fission promoting genes were concurrently downregulated in both infected cell lines and human samples. SARS-CoV-2 infection impeded autophagic flux either by upregulating GSK3B in lung cell lines or by downregulating autophagy genes, SNAP29, and lysosome acidification genes in human samples, contributing to increased viral replication. Therefore, drugs targeting lysosome acidification or autophagic flux could be tested as intervention strategies. Finally, age-stratified SARS-CoV-2-positive human data revealed impaired upregulation of chemokines, interferon-stimulated genes, and tripartite motif genes that are critical for antiviral signaling. Together, this analysis has revealed specific aspects of autophagic and mitochondrial function that are uniquely perturbed in SARS-CoV-2-infected host cells.

The metabolite acetyl-coenzyme A (acetyl-CoA) serves as an essential element for a wide range of cellular functions including adenosine triphosphate (ATP) production, lipid synthesis, and protein acetylation. Intracellular acetyl-CoA concentrations are associated with nutrient availability, but the mechanisms by which a cell responds to fluctuations in acetyl-CoA levels remain elusive. Here, we generate a cell system to selectively manipulate the nucleo-cytoplasmic levels of acetyl-CoA using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing and acetate supplementation of the culture media. Using this system and quantitative omics analyses, we demonstrate that acetyl-CoA depletion alters the integrity of the nucleolus, impairing ribosomal RNA synthesis and evoking the ribosomal protein-dependent activation of p53. This nucleolar remodeling appears to be mediated through the class IIa histone deacetylases (HDACs). Our findings highlight acetylation-mediated control of the nucleolus as an important hub linking acetyl-CoA fluctuations to cellular stress responses.

Complement, while serving to remove pathogens in the circulation, is also important for synergizing with inflammasomes to modulate CD4 T cell activation. Here the authors show that CD46, a complement receptor expressed only in humans, is essential for inducing optimal activation and effector functions of human CD8 T cells.

Chronic exposure to glucocorticoids (GCs) can lead to psychiatric complications through epigenetic mechanisms such as DNA methylation (DNAm). We sought to determine whether epigenetic changes in a peripheral tissue can serve as a surrogate for those in a relatively inaccessible tissue such as the brain. DNA extracted from the hippocampus and blood of mice treated with GCs or vehicle solution was assayed using a genome-wide DNAm platform (Methyl-Seq) to identify differentially methylated regions (DMRs) induced by GC treatment. We observed that ∼70% of the DMRs in both tissues lost methylation following GC treatment. Of the 3,095 DMRs that mapped to the same genes in both tissues, 1,853 DMRs underwent DNAm changes in the same direction. Interestingly, only 209 DMRs (

Autism spectrum disorders (ASDs) are characterized by deficits in three core behavioral domains: reciprocal social interactions, communication, and restricted interests and/or repetitive behaviors. Several hundreds of risk genes for autism have been identified, however, it remains a challenge to associate these genes with specific core behavioral deficits. In multiplex autism families, affected sibs often show significant differences in severity of individual core phenotypes. We hypothesize that a higher mutation burden contributes to a larger difference in the severity of specific core phenotypes between affected sibs. We tested this hypothesis on social behavioral deficits in autism. We sequenced synaptome genes (n = 1,886) in affected male sib-pairs (n = 274) in families from the Autism Genetics Research Exchange (AGRE) and identified rare (MAF ≤ 1%) and predicted functional variants. We selected affected sib-pairs with a large (≥10; n = 92 pairs) or a small (≤4; n = 108 pairs) difference in total cumulative Autism Diagnostic Interview-Revised (ADI-R) social scores (SOCT_CS). We compared burdens of unshared variants present only in sibs with severe social deficits and found a higher burden in SOCT_CS≥10 compared to SOCT_CS ≤ 4 (SOCT_CS≥10: 705.1 ± 16.2; SOCT_CS ≤ 4, 668.3 ± 9.0; p = 0.025). Unshared SOCT_CS≥10 genes only in sibs with severe social deficits are significantly enriched in the SFARI gene set. Network analyses of these genes using InWeb_IM, molecular signatures database (MSigDB), and GeNetMeta identified enrichment for phosphoinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) (Enrichment Score [eScore] p value = 3.36E−07; n = 8 genes) and Nerve growth factor (NGF) (eScore p value = 8.94E−07; n = 9 genes) networks. These studies support a key role for these signaling networks in social behavioral deficits and present a novel approach to associate risk genes and signaling networks with core behavioral domains in autism.

Oogenesis features an enormous increase in mitochondrial mass and mtDNA copy number, which are required to furnish mature eggs with an adequate supply of mitochondria and to curb the transmission of deleterious mtDNA variants. Quiescent in dividing germ cells, mtDNA replication initiates upon oocyte determination in the Drosophila ovary, which necessitates active mitochondrial respiration. However, the underlying mechanism for this dynamic regulation remains unclear. Here, we show that an feedforward insulin-Myc loop promotes mitochondrial respiration and biogenesis by boosting the expression of electron transport chain subunits and of factors essential for mtDNA replication and expression, and for the import of mitochondrial proteins. We further reveal that transient activation of JNK enhances the expression of the insulin receptor and initiates the insulin-Myc signaling loop. This signaling relay promotes mitochondrial biogenesis in the ovary, and thereby plays a role in limiting the transmission of deleterious mtDNA mutations. Our study demonstrates cellular mechanisms that couple mitochondrial biogenesis and inheritance with oocyte development.

Great effort is spent on developing therapies to improve the dire outcomes of those diagnosed with acute myeloid leukemia. The methods for quantifying response to therapeutic intervention have however lacked sensitivity. Patients achieving a complete remission as defined by conventional cytomorphological methods therefore remain at risk of subsequent relapse due to disease persistence. Improved risk stratification is possible based on tests designed to detect this residual leukemic burden (measurable residual disease). However, acute myeloid leukemia is a genetically diverse set of diseases, which has made it difficult to develop a single, highly reproducible, and sensitive assay for measurable residual disease. Here we present the development of a digital targeted RNA-sequencing-based approach designed to overcome these limitations by detecting all newly approved European LeukemiaNet molecular targets for measurable residual disease in acute myeloid leukemia in a single standardized assay. Iterative modifications and novel bioinformatics approaches resulted in a greater than 100-fold increase in performance compared with commercially available targeted RNA-sequencing approaches and a limit of detection as low as one leukemic cell in 100,000 cells measured, which is comparable to quantitative polymerase chain reaction analysis, the current gold standard for the detection of measurable residual disease. This assay, which can be customized and expanded, is the first demonstrated use of high-sensitivity RNA-sequencing for measurable residual disease detection in acute myeloid leukemia and could serve as a broadly applicable standardized tool.

The transcription factors TFE3 and TFEB cooperate to regulate autophagy induction and lysosome biogenesis in response to starvation. Here we demonstrate that DNA damage activates TFE3 and TFEB in a p53 and mTORC1 dependent manner. RNA-Seq analysis of TFEB/TFE3 double-knockout cells exposed to etoposide reveals a profound dysregulation of the DNA damage response, including upstream regulators and downstream p53 targets. TFE3 and TFEB contribute to sustain p53-dependent response by stabilizing p53 protein levels. In TFEB/TFE3 DKOs, p53 half-life is significantly decreased due to elevated Mdm2 levels. Transcriptional profiles of genes involved in lysosome membrane permeabilization and cell death pathways are dysregulated in TFEB/TFE3-depleted cells. Consequently, prolonged DNA damage results in impaired LMP and apoptosis induction. Finally, expression of multiple genes implicated in cell cycle control is altered in TFEB/TFE3 DKOs, revealing a previously unrecognized role of TFEB and TFE3 in the regulation of cell cycle checkpoints in response to stress.

Recently, the geographical origins of Ashkenazic Jews (AJs) and their native language Yiddish were investigated by applying the Geographic Population Structure (GPS) to a cohort of exclusively Yiddish-speaking and multilingual AJs. GPS localized most AJs along major ancient trade routes in northeastern Turkey adjacent to primeval villages with names that resemble the word “Ashkenaz.” These findings were compatible with the hypothesis of an Irano-Turko-Slavic origin for AJs and a Slavic origin for Yiddish and at odds with the Rhineland hypothesis advocating a Levantine origin for AJs and German origins for Yiddish. We discuss how these findings advance three ongoing debates concerning (1) the historical meaning of the term “Ashkenaz;” (2) the genetic structure of AJs and their geographical origins as inferred from multiple studies employing both modern and ancient DNA and original ancient DNA analyses; and (3) the development of Yiddish. We provide additional validation to the non-Levantine origin of AJs using ancient DNA from the Near East and the Levant. Due to the rising popularity of geo-localization tools to address questions of origin, we briefly discuss the advantages and limitations of popular tools with focus on the GPS approach. Our results reinforce the non-Levantine origins of AJs.

In the past few years, case-control studies of common diseases have shifted their focus from single genes to whole exomes. New sequencing technologies now routinely detect hundreds of thousands of sequence variants in a single study, many of which are rare or even novel. The limitation of classical single-marker association analysis for rare variants has been a challenge in such studies. A new generation of statistical methods for case-control association studies has been developed to meet this challenge. A common approach to association analysis of rare variants is the burden-style collapsing methods to combine rare variant data within individuals across or within genes. Here, we propose a new hybrid likelihood model that combines a burden test with a test of the position distribution of variants. In extensive simulations and on empirical data from the Dallas Heart Study, the new model demonstrates consistently good power, in particular when applied to a gene set (e.g., multiple candidate genes with shared biological function or pathway), when rare variants cluster in key functional regions of a gene, and when protective variants are present. When applied to data from an ongoing sequencing study of bipolar disorder (191 cases, 107 controls), the model identifies seven gene sets with nominal p-values < 0.05, of which one MAPK signaling pathway (KEGG) reaches trend-level significance after correcting for multiple testing.

High density oligonucleotide probe arrays have increasingly become an important tool in genomics studies. In organisms with incomplete genome sequence, one strategy for oligo probe design is to reduce the number of unique probes that target every non-redundant transcript through bioinformatic analysis and experimental testing. Here we adopted this strategy in making oligo probes for the earthworm Eisenia fetida, a species for which we have sequenced transcriptome-scale expressed sequence tags (ESTs). Our objectives were to identify unique transcripts as targets, to select an optimal and non-redundant oligo probe for each of these target ESTs, and to annotate the selected target sequences. We developed a streamlined and easy-to-follow approach to the design, validation and annotation of species-specific array probes. Four 244K-formatted oligo arrays were designed using eArray and were hybridized to a pooled E. fetida cRNA sample. We identified 63,541 probes with unsaturated signal intensities consistently above the background level. Target transcripts of these probes were annotated using several sequence alignment algorithms. Significant hits were obtained for 37,439 (59%) probed targets. We validated and made publicly available 63.5K oligo probes so the earthworm research community can use them to pursue ecological, toxicological, and other functional genomics questions. Our approach is efficient, cost-effective and robust because it (1) does not require a major genomics core facility; (2) allows new probes to be easily added and old probes modified or eliminated when new sequence information becomes available, (3) is not bioinformatics-intensive upfront but does provide opportunities for more in-depth annotation of biological functions for target genes; and (4) if desired, EST orthologs to the UniGene clusters of a reference genome can be identified and selected in order to improve the target gene specificity of designed probes. This approach is particularly applicable to organisms with a wealth of EST sequences but unfinished genome.