RESUMEN
To understand the neural basis of behavior, it is essential to measure spiking dynamics across many interacting brain regions. Although new technologies, such as Neuropixels probes, facilitate multi-regional recordings, significant surgical and procedural hurdles remain for these experiments to achieve their full potential. Here, we describe skull-shaped hemispheric implants enabling large-scale electrophysiology datasets (SHIELD). These 3D-printed skull-replacement implants feature customizable insertion holes, allowing dozens of cortical and subcortical structures to be recorded in a single mouse using repeated multi-probe insertions over many days. We demonstrate the procedure's high success rate, biocompatibility, lack of adverse effects on behavior, and compatibility with imaging and optogenetics. To showcase SHIELD's scientific utility, we use multi-probe recordings to reveal novel insights into how alpha rhythms organize spiking activity across visual and sensorimotor networks. Overall, this method enables powerful, large-scale electrophysiological experiments for the study of distributed neural computation.
RESUMEN
BACKGROUND AND AIMS: Extracellular vesicles (EVs) secreted by cardiosphere-derived cells exert immunomodulatory effects through the transmission of small non-coding RNAs. METHODS: The mechanism and role of yREX3, a small Y RNA abundant in EVs in myocardial injury, was investigated. RESULTS: yREX3 attenuates cardiac ischaemic injury by selective DNA methylation. Synthetic yREX3 encapsulated in lipid nanoparticles triggers broad transcriptomic changes in macrophages, localizes to the nucleus, and mediates epigenetic silencing of protein interacting with C kinase-1 (Pick1) through methylation of upstream CpG sites. Moreover, yREX3 interacts with polypyrimidine tract binding protein 3 (PTBP3) to methylate the Pick1 gene locus in a DNA methyltransferase-dependent manner. Suppression of Pick1 in macrophages potentiates Smad3 signalling and enhances efferocytosis, minimizing heart necrosis in rats with myocardial infarction. Adoptive transfer of Pick1-deficient macrophages recapitulates the cardioprotective effects of yREX3 in vivo. CONCLUSIONS: These findings highlight the role of a small Y RNA mined from EVs with a novel gene-methylating mechanism.
Asunto(s)
Vesículas Extracelulares , Macrófagos , Vesículas Extracelulares/metabolismo , Macrófagos/metabolismo , Animales , Humanos , Metilación de ADN , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Infarto del Miocardio/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Ratas , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , MasculinoRESUMEN
Multi-electrode arrays such as Neuropixels probes enable electrophysiological recordings from large populations of single neurons with high temporal resolution. By using such probes, the activity from functionally interacting, yet distinct, brain regions can be measured simultaneously by inserting multiple probes into the same subject. However, the use of multiple probes in small animals such as mice requires the removal of a sizable fraction of the skull, while also minimizing tissue damage and keeping the brain stable during the recordings. Here, we describe a step-by-step process designed to facilitate reliable recordings from up to six Neuropixels probes simultaneously in awake, head-fixed mice. The procedure involves four stages: the implantation of a headframe and a removable glass coverslip, the precise positioning of the Neuropixels probes at targeted points on the brain surface, the placement of a perforated plastic imaging window and the insertion of the probes into the brain of an awake mouse. The approach provides access to multiple brain regions and has been successfully applied across hundreds of mice. The procedure has been optimized for dense recordings from the mouse visual system, but it can be adapted for alternative recording configurations to target multiple probes in other brain areas. The protocol is suitable for users with experience in stereotaxic surgery in mice.
Asunto(s)
Neuronas , Vigilia , Ratones , Animales , Vigilia/fisiología , Neuronas/fisiología , Encéfalo/fisiología , Electrodos , Cabeza , Electrodos ImplantadosRESUMEN
The mammalian brain is composed of diverse neuron types that play different functional roles. Recent single-cell RNA sequencing approaches have led to a whole brain taxonomy of transcriptomically-defined cell types, yet cell type definitions that include multiple cellular properties can offer additional insights into a neuron's role in brain circuits. While the Patch-seq method can investigate how transcriptomic properties relate to the local morphological and electrophysiological properties of cell types, linking transcriptomic identities to long-range projections is a major unresolved challenge. To address this, we collected coordinated Patch-seq and whole brain morphology data sets of excitatory neurons in mouse visual cortex. From the Patch-seq data, we defined 16 integrated morpho-electric-transcriptomic (MET)-types; in parallel, we reconstructed the complete morphologies of 300 neurons. We unified the two data sets with a multi-step classifier, to integrate cell type assignments and interrogate cross-modality relationships. We find that transcriptomic variations within and across MET-types correspond with morphological and electrophysiological phenotypes. In addition, this variation, along with the anatomical location of the cell, can be used to predict the projection targets of individual neurons. We also shed new light on infragranular cell types and circuits, including cell-type-specific, interhemispheric projections. With this approach, we establish a comprehensive, integrated taxonomy of excitatory neuron types in mouse visual cortex and create a system for integrated, high-dimensional cell type classification that can be extended to the whole brain and potentially across species.
RESUMEN
Epigenetic clocks are DNA methylation-based chronological age prediction models that are commonly employed to study age-related biology. The difference between the predicted and observed age is often interpreted as a form of biological age acceleration, and many studies have measured the impact of environmental and disease-associated factors on epigenetic age. Most epigenetic clocks are fit using approaches that minimize the error between the predicted and observed chronological age, and as a result, they may not accurately model the impact of factors that moderate the relationship between the actual and epigenetic age. Here, we compare epigenetic clocks that are constructed using penalized regression methods to an evolutionary framework of epigenetic aging with the epigenetic pacemaker (EPM), which directly models DNA methylation as a function of a time-dependent epigenetic state. In simulations, we show that the value of the epigenetic state is impacted by factors such as age, sex, and cell-type composition. Next, in a dataset aggregated from previous studies, we show that the epigenetic state is also moderated by sex and the cell type. Finally, we demonstrate that the epigenetic state is also moderated by toxins in a study on polybrominated biphenyl exposure. Thus, we find that the pacemaker provides a robust framework for the study of factors that impact epigenetic age acceleration and that the effect of these factors may be obscured in traditional clocks based on linear regression models.
RESUMEN
Ecophysiologists have reported a range of relationships, including intrinsic trade-offs across and within species between plant relative growth rate in high resource conditions (RGR) vs adaptation to tolerate cold or arid climates, arising from trait-based mechanisms. Few studies have considered ecotypes within a species, in which the lack of a trade-off would contribute to a wide species range and resilience to climate change. For 15 ecotypes of Arabidopsis thaliana in a common garden we tested for associations between RGR vs adaptation to cold or dry native climates and assessed hypotheses for its mediation by 15 functional traits. Ecotypes native to warmer, drier climates had higher leaf density, leaf mass per area, root mass fraction, nitrogen per leaf area and carbon isotope ratio, and lower osmotic potential at full turgor. Relative growth rate was statistically independent of the climate of the ecotype native range and of individual functional traits. The decoupling of RGR and cold or drought adaptation in Arabidopsis is consistent with multiple stress resistance and avoidance mechanisms for ecotypic climate adaptation and would contribute to the species' wide geographic range and resilience as the climate changes.
Asunto(s)
Arabidopsis , Adaptación Fisiológica , Isótopos de Carbono , Ecotipo , Nitrógeno , Hojas de la PlantaRESUMEN
Background: Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and post-operative atrial fibrillation (POAF) is a major healthcare burden, contributing to an increased risk of stroke, kidney failure, heart attack and death. Genetic studies have identified associations with AF, but no molecular diagnostic exists to predict POAF based on pre-operative measurements. Such a tool would be of great value for perioperative planning to improve patient care and reduce healthcare costs. In this pilot study of epigenetic precision medicine in the perioperative period, we carried out bisulfite sequencing to measure DNA methylation status in blood collected from patients prior to cardiac surgery to identify biosignatures of POAF. Methods: We enrolled 221 patients undergoing cardiac surgery in this prospective observational study. DNA methylation measurements were obtained from blood samples drawn from awake patients prior to surgery. After controlling for clinical and methylation covariates, we analyzed DNA methylation loci in the discovery cohort of 110 patients for association with POAF. We also constructed predictive models for POAF using clinical and DNA methylation data. We subsequently performed targeted analyses of a separate cohort of 101 cardiac surgical patients to measure the methylation status solely of significant methylation loci in the discovery cohort. Results: A total of 47 patients in the discovery cohort (42.7%) and 43 patients in the validation cohort (42.6%) developed POAF. We identified 12 CpGs that were statistically significant in the discovery cohort after correcting for multiple hypothesis testing. Of these sites, 6 were amenable to targeted bisulfite sequencing and chr16:24640902 was statistically significant in the validation cohort. In addition, the methylation POAF prediction model had an AUC of 0.79 in the validation cohort. Conclusions: We have identified DNA methylation biomarkers that can predict future occurrence of POAF associated with cardiac surgery. This research demonstrates the use of precision medicine to develop models combining epigenomic and clinical data to predict disease.
RESUMEN
Unlike genomes, which are static throughout the lifespan of an organism, DNA methylomes are dynamic. To study these dynamics, we developed quantitative models that measure the effect of multiple factors on DNA methylomes including, age, sex, weight, and genetics. We conducted our study in canids, which prove to be an ideal species to assess epigenetic moderators due to their extreme variability in size and well-characterized genetic structure. We collected buccal swabs from 217 canids (207 domestic dogs and 10 grey wolves) and used targeted bisulphite sequencing to measure methylomes. We also measured genotypes at over one thousand single nucleotide polymorphisms (SNPs). As expected, we found that DNA methylomes are strongly associated with age, enabling the construction of epigenetic clocks. However, we also identify novel associations between methylomes and sex, weight, and sterilization status, leading to accurate models that predict these factors. Methylomes are also affected by genetics, and we observe multiple associations between SNP loci and methylated CpGs. Finally, we show that several factors moderate the relationship between epigenetic ages and real ages, such as body weight, which increases epigenetic ageing. In conclusion, we demonstrate that the plasticity of DNA methylomes is impacted by myriad genetics and physiological factors, and that DNA methylation biomarkers are accurate predictors of age, sex and sterilization status.
Asunto(s)
Metilación de ADN , Epigenoma , Animales , Perros , Epigenómica , Longevidad , Genotipo , Epigénesis GenéticaRESUMEN
In eukaryotic cells, intron lariats produced by the spliceosome contain a 2'5' phosphodiester linkage. The RNA lariat debranching enzyme, Dbr1, is the only enzyme known to hydrolyze this bond. Dbr1 is a member of the metallophosphoesterase (MPE) family of enzymes, and recent X-ray crystal structures and biochemistry data demonstrate that Dbr1 from Entamoeba histolytica uses combinations of Mn2+, Zn2+, and Fe2+ as enzymatic cofactors. Here, we examine the kinetic properties and metal dependence of the Dbr1 homolog from Saccharomyces cerevisiae (yDbr1). Elemental analysis measured stoichiometric quantities of Fe and Zn in yDbr1 purified following heterologous expression E. coli We analyzed the ability of Fe2+, Zn2+, and Mn2+ to reconstitute activity in metal-free apoenzyme. Purified yDbr1 was highly active, turning over substrate at 5.6 sec-1, and apo-yDbr1 reconstituted with Fe2+ was the most active species, turning over at 9.2 sec-1 We treated human lymphoblastoid cells with the iron-chelator deferoxamine and measured a twofold increase in cellular lariats. These data suggest that Fe is an important biological cofactor for Dbr1 enzymes.
Asunto(s)
Escherichia coli , Saccharomyces cerevisiae , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Intrones , Metales , ARN/química , ARN Nucleotidiltransferasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Immune cell-type composition changes with age, potentially weakening the response to infectious diseases. Profiling epigenetics marks of immune cells can help us understand the relationship with disease severity. We therefore leveraged a targeted DNA methylation method to study the differences in a cohort of pneumonia patients (both COVID-19 positive and negative) and unaffected individuals from peripheral blood.This approach allowed us to predict the pneumonia diagnosis with high accuracy (AUC = 0.92), and the PCR positivity to the SARS-CoV-2 viral genome with moderate, albeit lower, accuracy (AUC = 0.77). We were also able to predict the severity of pneumonia (PORT score) with an R2 = 0.69. By estimating immune cellular frequency from DNA methylation data, patients under the age of 65 positive to the SARS-CoV-2 genome (as revealed by PCR) showed an increase in T cells, and specifically in CD8+ cells, compared to the negative control group. Conversely, we observed a decreased frequency of neutrophils in the positive compared to the negative group. No significant difference was found in patients over the age of 65. The results suggest that this DNA methylation-based approach can be used as a cost-effective and clinically useful biomarker platform for predicting pneumonias and their severity.
Asunto(s)
COVID-19 , Neumonía , Humanos , SARS-CoV-2/genética , COVID-19/genética , Metilación de ADN , Neumonía/genética , BiomarcadoresRESUMEN
The epigenetic trajectory of DNA methylation profiles has a nonlinear relationship with time, reflecting rapid changes in DNA methylation early in life that progressively slow with age. In this study, we use pseudotime analysis to determine the functional form of these trajectories. Unlike epigenetic clocks that constrain the functional form of methylation changes with time, pseudotime analysis orders samples along a path, based on similarities in a latent dimension, to provide an unbiased trajectory. We show that pseudotime analysis can be applied to DNA methylation in human blood and brain tissue and find that it is highly correlated with the epigenetic states described by the Epigenetic Pacemaker. Moreover, we show that the pseudotime trajectory can be modeled with respect to time, using a sum of two exponentials, with coefficients that are close to the timescales of human age-associated mortality. Thus, for the first time, we can identify age-associated molecular changes that appear to track the exponential dynamics of mortality risk.
Asunto(s)
Metilación de ADN , Epigénesis Genética , Envejecimiento/genética , Encéfalo , Metilación de ADN/genética , Epigenómica/métodos , HumanosRESUMEN
We present a unique, extensive, and open synaptic physiology analysis platform and dataset. Through its application, we reveal principles that relate cell type to synaptic properties and intralaminar circuit organization in the mouse and human cortex. The dynamics of excitatory synapses align with the postsynaptic cell subclass, whereas inhibitory synapse dynamics partly align with presynaptic cell subclass but with considerable overlap. Synaptic properties are heterogeneous in most subclass-to-subclass connections. The two main axes of heterogeneity are strength and variability. Cell subclasses divide along the variability axis, whereas the strength axis accounts for substantial heterogeneity within the subclass. In the human cortex, excitatory-to-excitatory synaptic dynamics are distinct from those in the mouse cortex and vary with depth across layers 2 and 3.
Asunto(s)
Neocórtex/fisiología , Vías Nerviosas , Neuronas/fisiología , Sinapsis/fisiología , Transmisión Sináptica , Adulto , Animales , Conjuntos de Datos como Asunto , Potenciales Postsinápticos Excitadores , Femenino , Humanos , Potenciales Postsinápticos Inhibidores , Masculino , Ratones , Ratones Transgénicos , Modelos Neurológicos , Neocórtex/citología , Lóbulo Temporal/citología , Lóbulo Temporal/fisiología , Corteza Visual/citología , Corteza Visual/fisiologíaRESUMEN
Species that hibernate generally live longer than would be expected based solely on their body size. Hibernation is characterized by long periods of metabolic suppression (torpor) interspersed by short periods of increased metabolism (arousal). The torpor-arousal cycles occur multiple times during hibernation, and it has been suggested that processes controlling the transition between torpor and arousal states cause ageing suppression. Metabolic rate is also a known correlate of longevity; we thus proposed the 'hibernation-ageing hypothesis' whereby ageing is suspended during hibernation. We tested this hypothesis in a well-studied population of yellow-bellied marmots (Marmota flaviventer), which spend 7-8 months per year hibernating. We used two approaches to estimate epigenetic age: the epigenetic clock and the epigenetic pacemaker. Variation in epigenetic age of 149 samples collected throughout the life of 73 females was modelled using generalized additive mixed models (GAMM), where season (cyclic cubic spline) and chronological age (cubic spline) were fixed effects. As expected, the GAMM using epigenetic ages calculated from the epigenetic pacemaker was better able to detect nonlinear patterns in epigenetic ageing over time. We observed a logarithmic curve of epigenetic age with time, where the epigenetic age increased at a higher rate until females reached sexual maturity (two years old). With respect to circannual patterns, the epigenetic age increased during the active season and essentially stalled during the hibernation period. Taken together, our results are consistent with the hibernation-ageing hypothesis and may explain the enhanced longevity in hibernators.
Asunto(s)
Hibernación , Marmota , Animales , Epigénesis Genética , Femenino , Longevidad/genética , Marmota/genética , Marmota/metabolismo , Estaciones del AñoRESUMEN
DNA methylation modulates telomere function. In Arabidopsis thaliana, telomeric regions have a bimodal chromatin organization with unmethylated telomeres and methylated subtelomeres. To gain insight into this organization we have generated TAIR10-Tel, a modified version of the Arabidopsis reference genome with additional sequences at most chromosome ends. TAIR10-Tel has allowed us to analyse DNA methylation at nucleotide resolution level in telomeric regions. We have analysed the wild-type strain and mutants that encode inactive versions of all currently known relevant methyltransferases involved in cytosine methylation. These analyses have revealed that subtelomeric DNA methylation extends 1 to 2 kbp from Interstitial Telomeric Sequences (ITSs) that abut or are very near to telomeres. However, DNA methylation drops at the telomeric side of the telomere-subtelomere boundaries and disappears at the inner part of telomeres. We present a comprehensive and integrative model for subtelomeric DNA methylation that should help to decipher the mechanisms that govern the epigenetic regulation of telomeres. This model involves a complex network of interactions between methyltransferases and subtelomeric DNA sequences.
Asunto(s)
Arabidopsis , Metilación de ADN , Arabidopsis/genética , Epigénesis Genética , Metiltransferasas/genética , Telómero/genéticaRESUMEN
The Mendelian inheritance pattern of acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria is autosomal dominant, but the clinical phenotype is heterogeneous. Within the general population, penetrance is low, but among first-degree relatives of a symptomatic proband, penetrance is higher. These observations suggest that genetic factors, in addition to mutation of the specific enzyme of the biosynthetic pathway of heme, contribute to the clinical phenotype. Recent studies by others suggested that the genotype of the transporter protein ABCB6 contribute to the porphyria phenotype. Identifying the molecule(s) that are transported by ABCB6 has been problematic and has led to uncertainty with respect to how or if variants/mutants contribute to phenotypic heterogeneity. Knockout mouse models of Abcb6 have not provided a direction for investigation as homozygous knockout animals do not have a discrete phenotype. To address the proposed link between ABC6 genotype and porphyria phenotype, a large cohort of patients with acute hepatic porphyria and erythropoietic protoporphyria was analyzed. Our studies showed that ABCB6 genotype did not correlate with disease severity. Therefore, genotyping of ABCB6 in patients with acute hepatic porphyria and erythropoietic protoporphyria is not warranted.
Asunto(s)
Porfirias Hepáticas , Porfirias , Protoporfiria Eritropoyética , Transportadoras de Casetes de Unión a ATP , Animales , Humanos , Ratones , Ratones Noqueados , Porfobilinógeno Sintasa/deficiencia , Porfirias/genética , Porfirias Hepáticas/genética , Protoporfiria Eritropoyética/genéticaRESUMEN
Effective conservation and management of threatened wildlife populations require an accurate assessment of age structure to estimate demographic trends and population viability. Epigenetic aging models are promising developments because they estimate individual age with high accuracy, accurately predict age in related species, and do not require invasive sampling or intensive long-term studies. Using blood and biopsy samples from known age plains zebras (Equus quagga), we model epigenetic aging using two approaches: the epigenetic clock (EC) and the epigenetic pacemaker (EPM). The plains zebra EC has the potential for broad application within the genus Equus given that five of the seven extant wild species of the genus are threatened. We test the EC's ability to predict age in sister taxa, including two endangered species and the more distantly related domestic horse, demonstrating high accuracy in all cases. By comparing chronological and estimated age in plains zebras, we investigate age acceleration as a proxy of health status. An interaction between chronological age and inbreeding is associated with age acceleration estimated by the EPM, suggesting a cumulative effect of inbreeding on biological aging throughout life.
Asunto(s)
Distribución por Edad , Epigénesis Genética , Equidae/genética , Animales , Especies en Peligro de Extinción , Epigenómica , Equidae/fisiología , Caballos/fisiología , Modelos Genéticos , Dinámica Poblacional , Especificidad de la EspecieRESUMEN
The neocortex is disproportionately expanded in human compared with mouse1,2, both in its total volume relative to subcortical structures and in the proportion occupied by supragranular layers composed of neurons that selectively make connections within the neocortex and with other telencephalic structures. Single-cell transcriptomic analyses of human and mouse neocortex show an increased diversity of glutamatergic neuron types in supragranular layers in human neocortex and pronounced gradients as a function of cortical depth3. Here, to probe the functional and anatomical correlates of this transcriptomic diversity, we developed a robust platform combining patch clamp recording, biocytin staining and single-cell RNA-sequencing (Patch-seq) to examine neurosurgically resected human tissues. We demonstrate a strong correspondence between morphological, physiological and transcriptomic phenotypes of five human glutamatergic supragranular neuron types. These were enriched in but not restricted to layers, with one type varying continuously in all phenotypes across layers 2 and 3. The deep portion of layer 3 contained highly distinctive cell types, two of which express a neurofilament protein that labels long-range projection neurons in primates that are selectively depleted in Alzheimer's disease4,5. Together, these results demonstrate the explanatory power of transcriptomic cell-type classification, provide a structural underpinning for increased complexity of cortical function in humans, and implicate discrete transcriptomic neuron types as selectively vulnerable in disease.
Asunto(s)
Ácido Glutámico/metabolismo , Neocórtex/citología , Neocórtex/crecimiento & desarrollo , Neuronas/citología , Neuronas/metabolismo , Enfermedad de Alzheimer , Animales , Forma de la Célula , Colágeno/metabolismo , Electrofisiología , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Humanos , Lisina/análogos & derivados , Masculino , Ratones , Neocórtex/anatomía & histología , Neuronas/clasificación , Técnicas de Placa-Clamp , TranscriptomaRESUMEN
γδ T cells constitute a major portion of lymphocytes in the blood of both ruminants and swine. Subpopulations of swine γδ T cells have been distinguished by CD2 and CD8α expression. However, it was not clear if they have distinct expression profiles of their T-cell receptor (TCR) or WC1 genes. Identifying receptor expression will contribute to understanding the functional differences between these subpopulations and their contributions to immune protection. Here, we annotated three genomic assemblies of the swine TCRγ gene locus finding four gene cassettes containing C, J and V genes, although some haplotypes carried a null TRGC gene (TRGC4). Genes in the TRGC1 cassette were homologs of bovine TRGC5 cassette while the others were not homologous to bovine genes. Here we evaluated three principal populations of γδ T cells (CD2+/SWC5-, CD2-/SWC5+, and CD2-/SWC5-). Both CD2- subpopulations transcribed WC1 co-receptor genes, albeit with different patterns of gene expression but CD2+ cells did not. All subpopulations transcribed TCR genes from all four cassettes, although there were differences in expression levels. Finally, the CD2+ and CD2- γδ T-cell populations differed in their representation in various organs and tissues, presumably at least partially reflective of different ligand specificities for their receptors.
Asunto(s)
Bovinos/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Rumiantes/inmunología , Porcinos/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD2/metabolismo , Genes Codificadores de los Receptores de Linfocitos T/genética , Glicoproteínas de Membrana/metabolismoRESUMEN
BACKGROUND: Bisulfite sequencing is commonly used to measure DNA methylation. Processing bisulfite sequencing data is often challenging owing to the computational demands of mapping a low-complexity, asymmetrical library and the lack of a unified processing toolset to produce an analysis-ready methylation matrix from read alignments. To address these shortcomings, we have developed BiSulfite Bolt (BSBolt), a fast and scalable bisulfite sequencing analysis platform. BSBolt performs a pre-alignment sequencing read assessment step to improve efficiency when handling asymmetrical bisulfite sequencing libraries. FINDINGS: We evaluated BSBolt against simulated and real bisulfite sequencing libraries. We found that BSBolt provides accurate and fast bisulfite sequencing alignments and methylation calls. We also compared BSBolt to several existing bisulfite alignment tools and found BSBolt outperforms Bismark, BSSeeker2, BISCUIT, and BWA-Meth based on alignment accuracy and methylation calling accuracy. CONCLUSION: BSBolt offers streamlined processing of bisulfite sequencing data through an integrated toolset that offers support for simulation, alignment, methylation calling, and data aggregation. BSBolt is implemented as a Python package and command line utility for flexibility when building informatics pipelines. BSBolt is available at https://github.com/NuttyLogic/BSBolt under an MIT license.