Neuron-specific chromatin disruption at CpG islands and aging-related regions in Kabuki syndrome mice.

Many Mendelian developmental disorders caused by coding variants in epigenetic regulators have now been discovered. Epigenetic regulators are broadly expressed, and each of these disorders typically shows phenotypic manifestations from many different organ systems. An open question is whether the chromatin disruption-the root of the pathogenesis-is similar in the different disease-relevant cell types. This is possible in principle, because all these cell types are subject to effects from the same causative gene, which has the same kind of function (e.g., methylates histones) and is disrupted by the same germline variant. We focus on mouse models for Kabuki syndrome types 1 and 2 and find that the chromatin accessibility changes in neurons are mostly distinct from changes in B or T cells. This is not because the neuronal accessibility changes occur at regulatory elements that are only active in neurons. Neurons, but not B or T cells, show preferential chromatin disruption at CpG islands and at regulatory elements linked to aging. A sensitive analysis reveals that regulatory elements disrupted in B/T cells do show chromatin accessibility changes in neurons, but these are very subtle and of uncertain functional significance. Finally, we are able to identify a small set of regulatory elements disrupted in all three cell types. Our findings reveal the cellular-context-specific effect of variants in epigenetic regulators and suggest that blood-derived episignatures, although useful diagnostically, may not be well suited for understanding the mechanistic basis of neurodevelopment in Mendelian disorders of the epigenetic machinery.

Growth deficiency in a mouse model of Kabuki syndrome 2 bears mechanistic similarities to Kabuki syndrome 1.

Growth deficiency is a characteristic feature of both Kabuki syndrome 1 (KS1) and Kabuki syndrome 2 (KS2), Mendelian disorders of the epigenetic machinery with similar phenotypes but distinct genetic etiologies. We previously described skeletal growth deficiency in a mouse model of KS1 and further established that a Kmt2d-/- chondrocyte model of KS1 exhibits precocious differentiation. Here we characterized growth deficiency in a mouse model of KS2, Kdm6atm1d/+. We show that Kdm6atm1d/+ mice have decreased femur and tibia length compared to controls and exhibit abnormalities in cortical and trabecular bone structure. Kdm6atm1d/+ growth plates are also shorter, due to decreases in hypertrophic chondrocyte size and hypertrophic zone height. Given these disturbances in the growth plate, we generated Kdm6a-/- chondrogenic cell lines. Similar to our prior in vitro model of KS1, we found that Kdm6a-/- cells undergo premature, enhanced differentiation towards chondrocytes compared to Kdm6a+/+ controls. RNA-seq showed that Kdm6a-/- cells have a distinct transcriptomic profile that indicates dysregulation of cartilage development. Finally, we performed RNA-seq simultaneously on Kmt2d-/-, Kdm6a-/-, and control lines at Days 7 and 14 of differentiation. This revealed surprising resemblance in gene expression between Kmt2d-/- and Kdm6a-/- at both time points and indicates that the similarity in phenotype between KS1 and KS2 also exists at the transcriptional level.

Removing unwanted variation between samples in Hi-C experiments.

Hi-C data are commonly normalized using single sample processing methods, with focus on comparisons between regions within a given contact map. Here, we aim to compare contact maps across different samples. We demonstrate that unwanted variation, of likely technical origin, is present in Hi-C data with replicates from different individuals, and that properties of this unwanted variation change across the contact map. We present band-wise normalization and batch correction, a method for normalization and batch correction of Hi-C data and show that it substantially improves comparisons across samples, including in a quantitative trait loci analysis as well as differential enrichment across cell types.

Precision pharmacological reversal of strain-specific diet-induced metabolic syndrome in mice informed by epigenetic and transcriptional regulation.

Diet-related metabolic syndrome is the largest contributor to adverse health in the United States. However, the study of gene-environment interactions and their epigenomic and transcriptomic integration is complicated by the lack of environmental and genetic control in humans that is possible in mouse models. Here we exposed three mouse strains, C57BL/6J (BL6), A/J, and NOD/ShiLtJ (NOD), to a high-fat, high-carbohydrate diet, leading to varying degrees of metabolic syndrome. We then performed transcriptomic and genome-wide DNA methylation analyses for each strain and found overlapping but also highly divergent changes in gene expression and methylation upstream of the discordant metabolic phenotypes. Strain-specific pathway analysis of dietary effects revealed a dysregulation of cholesterol biosynthesis common to all three strains but distinct regulatory networks driving this dysregulation. This suggests a strategy for strain-specific targeted pharmacologic intervention of these upstream regulators informed by epigenetic and transcriptional regulation. As a pilot study, we administered the drug GW4064 to target one of these genotype-dependent networks, the farnesoid X receptor pathway, and found that GW4064 exerts strain-specific protection against dietary effects in BL6, as predicted by our transcriptomic analysis. Furthermore, GW4064 treatment induced inflammatory-related gene expression changes in NOD, indicating a strain-specific effect in its associated toxicities as well as its therapeutic efficacy. This pilot study demonstrates the potential efficacy of precision therapeutics for genotype-informed dietary metabolic intervention and a mouse platform for guiding this approach.

Quantification and statistical modeling of droplet-based single-nucleus RNA-sequencing data.

In complex tissues containing cells that are difficult to dissociate, single-nucleus RNA-sequencing (snRNA-seq) has become the preferred experimental technology over single-cell RNA-sequencing (scRNA-seq) to measure gene expression. To accurately model these data in downstream analyses, previous work has shown that droplet-based scRNA-seq data are not zero-inflated, but whether droplet-based snRNA-seq data follow the same probability distributions has not been systematically evaluated. Using pseudonegative control data from nuclei in mouse cortex sequenced with the 10x Genomics Chromium system and mouse kidney sequenced with the DropSeq system, we found that droplet-based snRNA-seq data follow a negative binomial distribution, suggesting that parametric statistical models applied to scRNA-seq are transferable to snRNA-seq. Furthermore, we found that the quantification choices in adapting quantification mapping strategies from scRNA-seq to snRNA-seq can play a significant role in downstream analyses and biological interpretation. In particular, reference transcriptomes that do not include intronic regions result in significantly smaller library sizes and incongruous cell type classifications. We also confirmed the presence of a gene length bias in snRNA-seq data, which we show is present in both exonic and intronic reads, and investigate potential causes for the bias.

Structure, Function, and Pharmacology of Glutamate Receptor Ion Channels.

Many physiologic effects of l-glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, are mediated via signaling by ionotropic glutamate receptors (iGluRs). These ligand-gated ion channels are critical to brain function and are centrally implicated in numerous psychiatric and neurologic disorders. There are different classes of iGluRs with a variety of receptor subtypes in each class that play distinct roles in neuronal functions. The diversity in iGluR subtypes, with their unique functional properties and physiologic roles, has motivated a large number of studies. Our understanding of receptor subtypes has advanced considerably since the first iGluR subunit gene was cloned in 1989, and the research focus has expanded to encompass facets of biology that have been recently discovered and to exploit experimental paradigms made possible by technological advances. Here, we review insights from more than 3 decades of iGluR studies with an emphasis on the progress that has occurred in the past decade. We cover structure, function, pharmacology, roles in neurophysiology, and therapeutic implications for all classes of receptors assembled from the subunits encoded by the 18 ionotropic glutamate receptor genes. SIGNIFICANCE STATEMENT: Glutamate receptors play important roles in virtually all aspects of brain function and are either involved in mediating some clinical features of neurological disease or represent a therapeutic target for treatment. Therefore, understanding the structure, function, and pharmacology of this class of receptors will advance our understanding of many aspects of brain function at molecular, cellular, and system levels and provide new opportunities to treat patients.

A functional genomics pipeline to identify high-value asthma and allergy CpGs in the human methylome.

BACKGROUND: DNA methylation of cytosines at cytosine-phosphate-guanine (CpG) dinucleotides (CpGs) is a widespread epigenetic mark, but genome-wide variation has been relatively unexplored due to the limited representation of variable CpGs on commercial high-throughput arrays. OBJECTIVES: To explore this hidden portion of the epigenome, this study combined whole-genome bisulfite sequencing with in silico evidence of gene regulatory regions to design a custom array of high-value CpGs. This study focused on airway epithelial cells from children with and without allergic asthma because these cells mediate the effects of inhaled microbes, pollution, and allergens on asthma and allergic disease risk. METHODS: This study identified differentially methylated regions from whole-genome bisulfite sequencing in nasal epithelial cell DNA from a total of 39 children with and without allergic asthma of both European and African ancestries. This study selected CpGs from differentially methylated regions, previous allergy or asthma epigenome-wide association studies (EWAS), or genome-wide association study loci, and overlapped them with functional annotations for inclusion on a custom Asthma&Allergy array. This study used both the custom and EPIC arrays to perform EWAS of allergic sensitization (AS) in nasal epithelial cell DNA from children in the URECA (Urban Environment and Childhood Asthma) birth cohort and using the custom array in the INSPIRE [Infant Susceptibility to Pulmonary Infections and Asthma Following RSV Exposure] birth cohort. Each CpG on the arrays was assigned to its nearest gene and its promotor capture Hi-C interacting gene and performed expression quantitative trait methylation (eQTM) studies for both sets of genes. RESULTS: Custom array CpGs were enriched for intermediate methylation levels compared to EPIC CpGs. Intermediate methylation CpGs were further enriched among those associated with AS and for eQTMs on both arrays. CONCLUSIONS: This study revealed signature features of high-value CpGs and evidence for epigenetic regulation of genes at AS EWAS loci that are robust to race/ethnicity, ascertainment, age, and geography.

Promoter CpG Density Predicts Downstream Gene Loss-of-Function Intolerance.

The aggregation and joint analysis of large numbers of exome sequences has recently made it possible to derive estimates of intolerance to loss-of-function (LoF) variation for human genes. Here, we demonstrate strong and widespread coupling between genic LoF intolerance and promoter CpG density across the human genome. Genes downstream of the most CpG-rich promoters (top 10% CpG density) have a 67.2% probability of being highly LoF intolerant, using the LOEUF metric from gnomAD. This is in contrast to 7.4% of genes downstream of the most CpG-poor (bottom 10% CpG density) promoters. Combining promoter CpG density with exonic and promoter conservation explains 33.4% of the variation in LOEUF, and the contribution of CpG density exceeds the individual contributions of exonic and promoter conservation. We leverage this to train a simple and easily interpretable predictive model that outperforms other existing predictors and allows us to classify 1,760 genes-which are currently unascertained in gnomAD-as highly LoF intolerant or not. These predictions have the potential to aid in the interpretation of novel variants in the clinical setting. Moreover, our results reveal that high CpG density is not merely a generic feature of human promoters but is preferentially encountered at the promoters of the most selectively constrained genes, calling into question the prevailing view that CpG islands are not subject to selection.

In vitro ADME characterization of a very potent 3-acylamino-2-aminopropionic acid-derived GluN2C-NMDA receptor agonist and its ester prodrugs.

The GluN2C subunit exists predominantly, but not exclusively in NMDA receptors within the cerebellum. Antagonists such as UBP1700 and positive allosteric modulators including PYD-106 and 3-acylamino-2-aminopropionic acid derivatives such as UA3-10 ((R)-2-amino-3-{[5-(2-bromophenyl)thiophen-2-yl]carboxamido}propionic acid) represent promising tool compounds to investigate the role of GluN2C-containing NMDA receptors in the signal transduction in the brain. However, due to its high polarity the bioavailability and CNS penetration of the amino acid UA3-10 are expected to be rather low. Herein, three ester prodrugs 12a-c of the NMDA receptor glycine site agonist UA3-10 were prepared and pharmacokinetically characterized. The esters 12a-c showed higher lipophilicity (higher logD 7.4 values) than the acid UA3-10 but almost the same binding at human serum albumin. The acid UA3-10 was rather stable upon incubation with mouse liver microsomes and NADPH, but the esters 12a-c were fast hydrolyzed to afford the acid UA3-10. Incubation with pig liver esterase and mouse serum led to rapid hydrolysis of the esters 12a-c. The isopropyl ester 12c showed a promising logD 7.4 value of 3.57 and the highest stability in the presence of pig liver esterase and mouse serum. These results demonstrate that ester prodrugs of UA3-10 can potentially afford improved bioavailability and CNS penetration.

Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing.

Probing epigenetic features on DNA has tremendous potential to advance our understanding of the phased epigenome. In this study, we use nanopore sequencing to evaluate CpG methylation and chromatin accessibility simultaneously on long strands of DNA by applying GpC methyltransferase to exogenously label open chromatin. We performed nanopore sequencing of nucleosome occupancy and methylome (nanoNOMe) on four human cell lines (GM12878, MCF-10A, MCF-7 and MDA-MB-231). The single-molecule resolution allows footprinting of protein and nucleosome binding, and determination of the combinatorial promoter epigenetic signature on individual molecules. Long-read sequencing makes it possible to robustly assign reads to haplotypes, allowing us to generate a fully phased human epigenome, consisting of chromosome-level allele-specific profiles of CpG methylation and chromatin accessibility. We further apply this to a breast cancer model to evaluate differential methylation and accessibility between cancerous and noncancerous cells.

Addressing the mean-correlation relationship in co-expression analysis.

Estimates of correlation between pairs of genes in co-expression analysis are commonly used to construct networks among genes using gene expression data. As previously noted, the distribution of such correlations depends on the observed expression level of the involved genes, which we refer to this as a mean-correlation relationship in RNA-seq data, both bulk and single-cell. This dependence introduces an unwanted technical bias in co-expression analysis whereby highly expressed genes are more likely to be highly correlated. Such a relationship is not observed in protein-protein interaction data, suggesting that it is not reflecting biology. Ignoring this bias can lead to missing potentially biologically relevant pairs of genes that are lowly expressed, such as transcription factors. To address this problem, we introduce spatial quantile normalization (SpQN), a method for normalizing local distributions in a correlation matrix. We show that spatial quantile normalization removes the mean-correlation relationship and corrects the expression bias in network reconstruction.

3D printed skulls in court - a benefit to stakeholders?

Forensic pathologists may use 3D prints as demonstrative aids when providing expert testimony in court of law, but the effects remain unclear despite many assumed benefits. In this qualitative study, the effects of using a 3D print, demonstrating a blunt force skull fracture, in court were explored by thematic analysis of interviews with judges, prosecutors, defence counsels, and forensic pathologists with the aim of improving the expert testimony. Five semi-structured focus groups and eight one-to-one interviews with a total of 29 stakeholders were transcribed ad verbatim and analysed using thematic analysis. The study found that a highly accurate 3D print of a skull demonstrated autopsy findings in detail and provided a quick overview, but sense of touch was of little benefit as the 3D print had different material characteristics than the human skull. Virtual 3D models were expected to provide all the benefits of 3D prints, be less emotionally confronting, and be logistically feasible. Both 3D prints and virtual 3D models were expected to be less emotionally confronting than autopsy photos. Regardless of fidelity, an expert witness was necessary to translate technical language and explain autopsy findings, and low-fidelity models may be equally suited as demonstrative aids. The court infrequently challenged the expert witnesses' conclusions and, therefore, rarely had a need for viewing autopsy findings in detail, therefore rarely needing a 3D print.

Differential Effects of Lipid Bilayers on αPSM Peptide Functional Amyloid Formation.

Phenol-soluble modulins (PSMs) are key virulence factors of S. aureus, and they comprise the structural scaffold of biofilm as they self-assemble into functional amyloids. They have been shown to interact with cell membranes as they display toxicity towards human cells through cell lysis, with αPSM3 being the most cytotoxic. In addition to causing cell lysis in mammalian cells, PSMs have also been shown to interact with bacterial cell membranes through antimicrobial effects. Here, we present a study on the effects of lipid bilayers on the aggregation mechanism of αPSM using chemical kinetics to study the effects of lipid vesicles on the aggregation kinetics and using circular dichroism (CD) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM) to investigate the corresponding secondary structure of the aggregates. We found that the effects of lipid bilayers on αPSM aggregation were not homogeneous between lipid type and αPSM peptides, although none of the lipids caused changes in the dominating aggregation mechanism. In the case of αPSM3, all types of lipids slowed down aggregation to a varying degree, with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) having the most pronounced effect. For αPSM1, lipids had opposite effects, where DOPC decelerated aggregation and lipopolysaccharide (LPS) accelerated the aggregation, while 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DOPG) had no effect. For αPSM4, both DOPG and LPS accelerated the aggregation, but only at high concentration, while DOPC showed no effect. None of the lipids was capable of inducing aggregation of αPSM2. Our data reveal a complex interaction pattern between PSMs peptides and lipid bilayers that causes changes in the aggregation kinetics by affecting different kinetic parameters along with only subtle changes in morphology.

A metabolomics approach identified toxins associated with uremic symptoms in advanced chronic kidney disease.

Uremic symptoms are common in patients with advanced chronic kidney disease, but the toxins that cause these symptoms are unknown. To evaluate this, we performed a cross-sectional study of the 12 month post-randomization follow-up visit of Modification of Diet in Renal Disease (MDRD) participants reporting uremic symptoms who also had available stored serum. We quantified 1,163 metabolites by liquid chromatography-tandem mass spectrometry. For each uremic symptom, we calculated a score as the severity multiplied by the number of days the symptom was experienced. We analyzed the associations of the individual symptom scores with metabolites using linear models with empirical Bayesian inference, adjusted for multiple comparisons. Among 695 participants, the mean measured glomerular filtration rate (mGFR) was 28 mL/min/1.73 m2. Uremic symptoms were more common in the subgroup of 214 patients with an mGFR under 20 mL/min/1.73 m2 (mGFR under 20 subgroup) than in the full group. For all metabolites with significant associations, the direction of the association was concordant in the full group and the subgroup. For gastrointestinal symptoms (bad taste, loss of appetite, nausea, and vomiting), eleven metabolites were associated with symptoms. For neurologic symptoms (decreased alertness, falling asleep during the day, forgetfulness, lack of pep and energy, and tiring easily/weakness), seven metabolites were associated with symptoms. Associations were consistent across sensitivity analyses. Thus, our proof-of-principle study demonstrates the potential for metabolomics to understand metabolic pathways associated with uremic symptoms. Larger, prospective studies with external validation are needed.

Coexpression patterns define epigenetic regulators associated with neurological dysfunction.

Coding variants in epigenetic regulators are emerging as causes of neurological dysfunction and cancer. However, a comprehensive effort to identify disease candidates within the human epigenetic machinery (EM) has not been performed; it is unclear whether features exist that distinguish between variation-intolerant and variation-tolerant EM genes, and between EM genes associated with neurological dysfunction versus cancer. Here, we rigorously define 295 genes with a direct role in epigenetic regulation (writers, erasers, remodelers, readers). Systematic exploration of these genes reveals that although individual enzymatic functions are always mutually exclusive, readers often also exhibit enzymatic activity (dual-function EM genes). We find that the majority of EM genes are very intolerant to loss-of-function variation, even when compared to the dosage sensitive transcription factors, and we identify 102 novel EM disease candidates. We show that this variation intolerance is driven by the protein domains encoding the epigenetic function, suggesting that disease is caused by a perturbed chromatin state. We then describe a large subset of EM genes that are coexpressed within multiple tissues. This subset is almost exclusively populated by extremely variation-intolerant genes and shows enrichment for dual-function EM genes. It is also highly enriched for genes associated with neurological dysfunction, even when accounting for dosage sensitivity, but not for cancer-associated EM genes. Finally, we show that regulatory regions near epigenetic regulators are genetically important for common neurological traits. These findings prioritize novel disease candidate EM genes and suggest that this coexpression plays a functional role in normal neurological homeostasis.

Hyperpolarized 13 C MRI Reveals Large Changes in Pyruvate Metabolism During Digestion in Snakes.

PURPOSE: Hyperpolarized 13 C MRI is a powerful technique to study dynamic metabolic processes in vivo; but it has predominantly been used in mammals, mostly humans, pigs, and rodents. METHODS: In the present study, we use this technique to characterize the metabolic fate of hyperpolarized [1-13 C]pyruvate in Burmese pythons (Python bivittatus), a large species of constricting snake that exhibits a four- to tenfold rise in metabolism and large growth of the visceral organs within 24-48 h of ingestion of their large meals. RESULTS: We demonstrate a fivefold elevation of the whole-body lactate-to-pyruvate ratio in digesting snakes, pointing to a large rise in lactate production from pyruvate. Consistent with the well-known metabolic stimulation of digestion, measurements of mitochondrial respiration in hepatocytes in vitro indicate a marked postprandial upregulation of mitochondrial respiration. We observed that a low SNR of the hyperpolarized 13 C produced metabolites in the python, and this lack of signal was possibly due to the low metabolism of reptiles compared with mammals, preventing quantification of alanine and bicarbonate production with the experimental setup used in this study. Spatial quantification of the [1-13 C]lactate was only possible in postprandial snakes (with high metabolism), where a statistically significant difference between the heart and liver was observed. CONCLUSION: We confirm the large postprandial rise in the wet mass of most visceral organs, except for the heart, and demonstrated that it is possible to image the [1-13 C]pyruvate uptake and intracellular conversion to [1-13 C]lactate in ectothermic animals.

Intrinsic Heart Regeneration in Adult Vertebrates May be Strictly Limited to Low-Metabolic Ectotherms.

The heart has a high-metabolic rate, and its "around-the-clock" vital role to sustain life sets it apart in a regenerative setting from other organs and appendages. The landscape of vertebrate species known to perform intrinsic heart regeneration is strongly biased toward ectotherms-for example, fish, salamanders, and embryonic/neonatal ectothermic mammals. It is hypothesized that intrinsic heart regeneration is exclusively limited to the low-metabolic hearts of ectotherms. The biomedical field of regenerative medicine seeks to devise biologically inspired regenerative therapies to diseased human hearts. Falsification of the ectothermy dependency for heart regeneration hypothesis may be a crucial prerequisite to meaningfully seek inspiration in established ectothermic regenerative animal models. Otherwise, engineering approaches to construct artificial heart components may constitute a more viable path toward regenerative therapies. A more strict definition of regenerative phenomena is generated and several testable sub-hypotheses and experimental avenues are put forward to elucidate the link between heart regeneration and metabolism. Also see the video abstract here https://youtu.be/fZcanaOT5z8.

Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping.

There is an urgent and unprecedented need for sensitive and high-throughput molecular diagnostic tests to combat the SARS-CoV-2 pandemic. Here we present a generalized version of the RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) assay, called "capture RASL-seq" (cRASL-seq), which enables highly sensitive (down to ~1-100 pfu/ml or cfu/ml) and highly multiplexed (up to ~10,000 target sequences) detection of pathogens. Importantly, cRASL-seq analysis of COVID-19 patient nasopharyngeal (NP) swab specimens does not involve nucleic acid purification or reverse transcription, steps that have introduced supply bottlenecks into standard assay workflows. Our simplified protocol additionally enables the direct and efficient genotyping of selected, informative SARS-CoV-2 polymorphisms across the entire genome, which can be used for enhanced characterization of transmission chains at population scale and detection of viral clades with higher or lower virulence. Given its extremely low per-sample cost, simple and automatable protocol and analytics, probe panel modularity, and massive scalability, we propose that cRASL-seq testing is a powerful new technology with the potential to help mitigate the current pandemic and prevent similar public health crises.

Optimizing hemodynamic function during cardiopulmonary resuscitation.

PURPOSE OF REVIEW: The purpose of this narrative review is to provide an update on hemodynamics during cardiopulmonary resuscitation (CPR) and to describe emerging therapies to optimize perfusion. RECENT FINDINGS: Cadaver studies have shown large inter-individual variations in blood distribution and anatomical placement of the heart during chest compressions. Using advanced CT techniques the studies have demonstrated atrial and slight right ventricular compression, but no direct compression of the left ventricle. A hemodynamic-directed CPR strategy may overcome this by allowing individualized hand-placement, drug dosing, and compression rate and depth. Through animal studies and one clinical before-and-after study head-up CPR has shown promising results as a potential strategy to improve cerebral perfusion. Two studies have demonstrated that placement of an endovascular balloon occlusion in the aorta (REBOA) can be performed during ongoing CPR. SUMMARY: Modern imaging techniques may help increase our understanding on the mechanism of forward flow during CPR. This could provide new information on how to optimize perfusion. Head-up CPR and the use of REBOA during CPR are novel methods that might improve cerebral perfusion during CPR; both techniques do, however, still await clinical testing.

Analyzing whole genome bisulfite sequencing data from highly divergent genotypes.

In the study of DNA methylation, genetic variation between species, strains or individuals can result in CpG sites that are exclusive to a subset of samples, and insertions and deletions can rearrange the spatial distribution of CpGs. How to account for this variation in an analysis of the interplay between sequence variation and DNA methylation is not well understood, especially when the number of CpG differences between samples is large. Here, we use whole-genome bisulfite sequencing data on two highly divergent mouse strains to study this problem. We show that alignment to personal genomes is necessary for valid methylation quantification. We introduce a method for including strain-specific CpGs in differential analysis, and show that this increases power. We apply our method to a human normal-cancer dataset, and show this improves accuracy and power, illustrating the broad applicability of our approach. Our method uses smoothing to impute methylation levels at strain-specific sites, thereby allowing strain-specific CpGs to contribute to the analysis, while accounting for differences in the spatial occurrences of CpGs. Our results have implications for joint analysis of genetic variation and DNA methylation using bisulfite-converted DNA, and unlocks the use of personal genomes for addressing this question.