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Artificial Sweeteners Induce Glucose Intolerance by Altering the Gut Microbiota
Abstract and Figures
Non-caloric artificial sweeteners (NAS) are among the most widely used food additives worldwide, regularly consumed by lean and obese individuals alike. NAS consumption is considered safe and beneficial owing to their low caloric content, yet supporting scientific data remain sparse and controversial. Here we demonstrate that consumption of commonly used NAS formulations drives the development of glucose intolerance through induction of compositional and functional alterations to the intestinal microbiota. These NAS-mediated deleterious metabolic effects are abrogated by antibiotic treatment, and are fully transferrable to germ-free mice upon faecal transplantation of microbiota configurations from NAS-consuming mice, or of microbiota anaerobically incubated in the presence of NAS. We identify NAS-altered microbial metabolic pathways that are linked to host susceptibility to metabolic disease, and demonstrate similar NAS-induced dysbiosis and glucose intolerance in healthy human subjects. Collectively, our results link NAS consumption, dysbiosis and metabolic abnormalities, thereby calling for a reassessment of massive NAS usage.
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... Low-calorie sweeteners, also known as non-nutritive sweeteners, have been used for decades to replace sugar and reduce the caloric content of foods and beverages while maintaining the sweet taste (1). The consumption of non-nutritive sweeteners has raised concerns about potential detrimental effects of long-term intake which have been demonstrated in some rodent studies (2)(3)(4)(5) but less consistently in human studies (4,(6)(7)(8). Aspartame and stevia are two commonly used low-calorie sweeteners whose metabolism has been investigated in different species (3,(9)(10)(11)(12)(13)(14). ...
... Non-nutritive sweeteners are non-toxic to human adults (3,9,11) but detrimental effects, including disrupted gut microbiota, impaired glucose homeostasis and higher risk of obesity have been observed in offspring of rodents consuming non-nutritive sweeteners and infants of pregnant women consuming beverages with non-nutritive sweeteners (2,3,5,7,9). We recently reported that maternal consumption of aspartame and stevia altered the expression of genes related to the mesolimbic reward system in 3-week and 18-week old rat offspring, and altered gut microbiota in the 3-week old offspring (9). ...
... Artificial sweeteners induce compositional and functional alterations in gut microbiota of human and rats (5,8,9). These shifts in gut microbiota have been linked to the development of obesity-related glucose intolerance which is transferable to germ-free mice through fecal microbiota transplantation (5,9,37). ...
To identify possible mechanisms by which maternal consumption of non-nutritive sweeteners increases obesity risk in offspring, we reconstructed the major alterations in the cecal microbiome of 3-week-old offspring of obese dams consuming high fat/sucrose (HFS) diet with or without aspartame (5–7 mg/kg/day) or stevia (2–3 mg/kg/day) by shotgun metagenomic sequencing (n = 36). High throughput 16S rRNA gene sequencing (n = 105) was performed for dams, 3- and 18-week-old offspring. Maternal consumption of sweeteners altered cecal microbial composition and metabolism of propionate/lactate in their offspring. Offspring daily body weight gain, liver weight and body fat were positively correlated to the relative abundance of key microbes and enzymes involved in succinate/propionate production while negatively correlated to that of lactose degradation and lactate production. The altered propionate/lactate production in the cecum of weanlings from aspartame and stevia consuming dams implicates an altered ratio of dietary carbohydrate digestion, mainly lactose, in the small intestine vs. microbial fermentation in the large intestine. The reconstructed microbiome alterations could explain increased offspring body weight and body fat. This study demonstrates that intense sweet tastants have a lasting and intergenerational effect on gut microbiota, microbial metabolites and host health.
... A well-functioning, healthy intestine is the key to convert feed into fish biomass efficiently. It is now well established that the intestinal microbiota is, in various ways, closely connected to intestinal function and health [17][18][19][20][21]. Diet is arguably one of the most important environmental factors shaping intestinal microbiota [22][23][24]. Different dietary components may selectively induce compositional and functional alterations of the intestinal microbiota, which in turn could inflict important implications on the host health and disease resistance [19,[24][25][26]. ...
... It is now well established that the intestinal microbiota is, in various ways, closely connected to intestinal function and health [17][18][19][20][21]. Diet is arguably one of the most important environmental factors shaping intestinal microbiota [22][23][24]. Different dietary components may selectively induce compositional and functional alterations of the intestinal microbiota, which in turn could inflict important implications on the host health and disease resistance [19,[24][25][26]. ...
Background: Being part of fish's natural diets, insects have become a practical alternative feed ingredient for aquaculture. While nutritional values of insects have been extensively studied in various fish species, their impact on the fish microbiota remains to be fully explored. In an 8-week freshwater feeding trial, Atlantic salmon (Salmo salar) were fed either a commercially relevant reference diet or an insect meal diet wherein black soldier fly (Hermetia illucens) larvae meal comprised 60% of total ingredients. Microbiota of digesta and mucosa origin from the proximal and distal intestine were collected and profiled along with feed and water samples.
Results: The insect meal diet markedly modulated the salmon intestinal microbiota. Salmon fed the insect meal diet showed similar or lower alpha-diversity indices in the digesta but higher alpha-diversity indices in the mucosa. A group of bacterial genera, dominated by members of the Bacillaceae family, was enriched in salmon fed the insect meal diet, which confirms our previous findings in a seawater feeding trial. We also found that microbiota in the intestine closely resembled that of the feeds but was distinct from the water microbiota. Notably, bacterial genera associated with the diet effects were also present in the feeds.
Conclusions: We conclude that salmon fed the insect meal diets show consistent changes in the intestinal micro-biota. The next challenge is to evaluate the extent to which these alterations are attributable to feed microbiota and dietary nutrients, and what these changes mean for fish physiology and health.
... Diferentes estudios han planteado que los edulcorantes artificiales como la sucralosa, la sacarina, el aspartame y el acesulfame potásico provocan desbalances en el microbioma intestinal (Dudek-Wicher et al., 2018). En una investigación con roedores, cinco semanas de uso de sacarina produjeron intolerancia a la glucosa como consecuencia de trastornos en el microbioma intestinal, caracterizados por un repunte en la proporción de Firmicutes/Bacteroidetes -similar a lo registrado en personas obesas-y alteraciones en las vías metabólicas microbianas asociadas con la susceptibilidad a enfermedades metabólicas en el organismo huésped (Suez et al., 2014). De igual modo, se reportaron correlaciones positivas entre el consumo a largo plazo de edulcorantes y la subida del peso, relación cintura-cadera, niveles elevados de glucosa en ayunas e intolerancia a la glucosa en 381 sujetos no diabéticos (Suez et al., 2014). ...
... En una investigación con roedores, cinco semanas de uso de sacarina produjeron intolerancia a la glucosa como consecuencia de trastornos en el microbioma intestinal, caracterizados por un repunte en la proporción de Firmicutes/Bacteroidetes -similar a lo registrado en personas obesas-y alteraciones en las vías metabólicas microbianas asociadas con la susceptibilidad a enfermedades metabólicas en el organismo huésped (Suez et al., 2014). De igual modo, se reportaron correlaciones positivas entre el consumo a largo plazo de edulcorantes y la subida del peso, relación cintura-cadera, niveles elevados de glucosa en ayunas e intolerancia a la glucosa en 381 sujetos no diabéticos (Suez et al., 2014). ...
Obesity is a multifactorial trait provoked by the interaction of biological, environmental, psychosocial, and socioeconomic factors. Proposal: The goal of the present review is to discuss the role of ultra-processed and highly palatable foods (UPHP) in the development of the obesity epidemic through an exploratory-descriptive review and to present some suggestions for controlling its consumption. Arguments for discussion: UPHP are energy dense foods with high contents of fat and sugar. UPHP are formulated with many industrial additives used for enhancing flavor, shelf life, and the stability of their components. UPHP used to contain diverse chemicals known as endocrine disruptors (EDC), which are transferred from packaging to foods, with bisphenol A and phthalates as the most common EDC. The EDC disrupt different hormonal signaling pathways affecting the metabolism of the adipose tissue and other endocrine systems. The overconsumption of UPHP induces neuroplastic changes in the brain reward system that increases their consumption, leading to body fat accumulation. In addition, the overconsumption of UPHP alters the composition of the intestinal microbiome (dysbiosis), which is associated with the development of obesity. Conclusions: The overconsumption of UPHP increases the risk of obesity and its related chronic, non-communicable diseases, especially when consumption initiates during early life. To counteract this problem, we proposed the following actions: changing the structure of the market-food basket, incorporating regulations to reduce the UPHP supply in and around educational centers, creating new taxes upon UPHP, and strengthening the research regarding obesity, and the effects of UPHP and EDC.
... High-fat, high-sugar and low-fibre diets reduce the richness of gut microbiota and promote the expansion of pathogenic species [16][17][18]. Food additives, which are highly prevalent in the Western diet, also impair gut homeostasis and promote intestinal inflammation [19][20][21][22][23]. ...
Intestinal dysbiosis has been widely documented in inflammatory bowel diseases (IBDs) and is thought to influence the onset and perpetuation of gut inflammation. However, it remains unclear whether such bacterial changes rely in part on the modification of an IBD-associated lifestyle (e.g., smoking and physical activity) and diet (e.g., rich in dairy products, cereals, meat and vegetables). In this study, we investigated the impact of these habits, which we defined as confounders and covariates, on the modulation of intestinal taxa abundance and diversity in IBD patients. 16S rRNA gene sequence analysis was performed using genomic DNA extracted from the faecal samples of 52 patients with Crohn’s disease (CD) and 58 with ulcerative colitis (UC), which are the two main types of IBD, as well as 42 healthy controls (HC). A reduced microbial diversity was documented in the IBD patients compared with the HC. Moreover, we identified specific confounders and covariates that influenced the association between some bacterial taxa and disease extent (in UC patients) or behaviour (in CD patients) compared with the HC. In particular, a PERMANOVA stepwise regression identified the variables “age”, “eat yogurt at least four days per week” and “eat dairy products at least 4 days per week” as covariates when comparing the HC and patients affected by ulcerative proctitis (E1), left-sided UC (distal UC) (E2) and extensive UC (pancolitis) (E3). Instead, the variables “age”, “gender”, “eat meat at least four days per week” and “eat bread at least 4 days per week” were considered as covariates when comparing the HC with the CD patients affected by non-stricturing, non-penetrating (B1), stricturing (B2) and penetrating (B3) diseases. Considering such variables, our analysis indicated that the UC extent differentially modulated the abundance of the Bifidobacteriaceae, Rikenellaceae, Christensenellaceae, Marinifilaceae, Desulfovibrionaceae, Lactobacillaceae, Streptococcaceae and Peptostreptococcaceae families, while the CD behaviour influenced the abundance of Christensenellaceae, Marinifilaceae, Rikenellaceae, Ruminococcaceae, Barnesiellaceae and Coriobacteriaceae families. In conclusion, our study indicated that some covariates and confounders related to an IBD-associated lifestyle and dietary habits influenced the intestinal taxa diversity and relative abundance in the CD and UC patients compared with the HC. Indeed, such variables should be identified and excluded from the analysis to characterize the bacterial families whose abundance is directly modulated by IBD status, as well as disease extent or behaviour.
... Sweeteners such as sucralose may promote and exacerbate inflammation by altering the microbiota composition when consumed over six months (Bian et al. 2017). Similarly, exposure to saccharin, sucralose, or aspartame in mice altered the microbiota composition and function, leading to glucose intolerance, suggesting these can be risk factors for metabolic diseases such as diabetes (Suez et al. 2014). Contrary to these findings, saccharin intake altered microbiome composition and reduced inflammation in mice with acute and chronic colitis (Sunderhauf et al. 2020). ...
Diet exerts a major influence upon host immune function and the gastrointestinal microbiota. Although components of the human diet (including carbohydrates, fats, and proteins) are essential sources of nutrition for the host, they also influence immune function directly through interaction with innate and cell-mediated immune regulatory mechanisms. Regulation of the microbiota community structure also provides a mechanism by which food components influence host immune regulatory processes. Here, we consider the complex interplay between components of the modern (Western) diet, the microbiota, and host immunity in the context of obesity and metabolic disease, inflammatory bowel disease, and infection.
Expected final online publication date for the Annual Review of Food Science and Technology, Volume 13 is March 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Traditional medicine systems around the globe, like Unani, Ayurveda and traditional Chinese medicine, include a number of sugar-based formulations, which contain a large amount of saccharide-containing sweetener, such as honey, sucrose or jaggery. With pervasive lifestyle disorders throughout the world, there have been discussions to consider alternative sweetening agents. Here, from the perspective of Unani medicine, we discuss how the saccharide-based sweeteners may be an essential component of these traditional preparations, like electuaries, which may be deprived of their bioactivities without these saccharides. With contemporary researches, it is known that apart from their own therapeutic effects, saccharides also form deep eutectic solvents which help in enhancing the bioactivity of other ingredients present in crude drugs. In addition, they provide energy for fermentation which is essential for biotransformation of compounds. Interestingly, the sugars also increase the shelf-life of these compound drugs and act as natural preservatives. On the basis of this review, we strongly believe that saccharide-based sweeteners are an essential component of traditional medicines and not merely an excipient.
The gut microbiome serves as a critical regulator of human physiology. The gut community is influenced by various external factors, mainly diet, pharmaceuticals, stress, exercise, etc. The gut microbiome can be of benefit or harm to the host depending on the metabolites produced. It has been shown that the gut microbiome also plays a significant role on the host metabolism. The alteration of gut microbial composition and reduction of its diversity contribute to the incidence of metabolic diseases. On the other hand, manipulation of gut microbial ecology through balancing some specific types of bacteria or enhancing the number of good bacteria can potentially prevent and serve as a therapeutic approach against metabolic related diseases. This chapter reviews how the gut microbiome interacts with the host, particularly in regulation of metabolism. The involvement of the gut microbiome with the development of metabolic diseases is highlighted. The modulation of the gut microbiome as a target source for prevention of metabolic diseases and possible mechanisms have been reviewed.
Background: Saccharin is a common artificial sweetener and a bona fide ligand for sweet taste receptors (STR). STR can regulate insulin secretion in beta cells, so we investigated whether saccharin can stimulate insulin secretion dependent on STR and the activation of phospholipase C (PLC) signaling. Methods: We performed in vivo and in vitro approaches in mice and cells with loss-of-function of STR signaling and specifically assessed the involvement of a PLC signaling cascade using real-time biosensors and calcium imaging. Results: We found that the ingestion of a physiological amount of saccharin can potentiate insulin secretion dependent on STR. Similar to natural sweeteners, saccharin triggers the activation of the PLC signaling cascade, leading to calcium influx and the vesicular exocytosis of insulin. The effects of saccharin also partially require transient receptor potential cation channel M5 (TRPM5) activity. Conclusions: Saccharin ingestion may transiently potentiate insulin secretion through the activation of the canonical STR signaling pathway. These physiological effects provide a framework for understanding the potential health impact of saccharin use and the contribution of STR in peripheral tissues.
Due to the strong polarity and difficult in biodegradation, acesulfame (ACE) and saccharin (SAC) are difficult to treat effectively in water environment. Herein, a series of ACE and SAC adsorption were investigated by the zinc-based MOFs (ZIF-8, MAF-6, MOF-5 and Zn-MOF-74) derived porous carbons (MPCs). The porous carbon derived from MOF-5 (MPCs-M5) and Zn-MOF-74 (MPCs-M74) of zinc-based carboxylate MOFs exhibited large surface area (2774.7 m²/g and 2447.3 m²/g), high mesoporosity (94.57% and 95.66%) and equipotential point value (7.85 and 9.82). The pseudo-second-order kinetic model and Freundlich adsorption model were fitted well to explore adsorption on the MPCs-M5 and MPCs-M74, respectively. Moreover, the MPCs-M5 and MPCs-M74 showed notable adsorption capacity of ACE (96.4 mg/g and 88.6 mg/g) and SAC (117.3 mg/g and 113.4 mg/g), respectively, which were the highest value than the previous reports. Furthermore, the excellent adsorption performance of MPCs-M5 and MPCs-M74 were also contributed by multi-faceted synergy of electrostatic, H-bond, and π-π interactions. Therefore, the above two samples of the hierarchical porous carbon prepared by self-activating zinc-based carboxylate MOFs with high oxygen content are recommended as efficient adsorbents for artificial sweeteners adsorption from water environment.
Assessment and characterization of gut microbiota has become a major research area in human disease, including type 2 diabetes, the most prevalent endocrine disease worldwide. To carry out analysis on gut microbial content in patients with type 2 diabetes, we developed a protocol for a metagenome-wide association study (MGWAS) and undertook a two-stage MGWAS based on deep shotgun sequencing of the gut microbial DNA from 345 Chinese individuals. We identified and validated approximately 60,000 type-2-diabetes-associated markers and established the concept of a metagenomic linkage group, enabling taxonomic species-level analyses. MGWAS analysis showed that patients with type 2 diabetes were characterized by a moderate degree of gut microbial dysbiosis, a decrease in the abundance of some universal butyrate-producing bacteria and an increase in various opportunistic pathogens, as well as an enrichment of other microbial functions conferring sulphate reduction and oxidative stress resistance. An analysis of 23 additional individuals demonstrated that these gut microbial markers might be useful for classifying type 2 diabetes.
Long-term dietary intake influences the structure and activity of the trillions of microorganisms residing in the human gut, but it remains unclear how rapidly and reproducibly the human gut microbiome responds to short-term macronutrient change. Here we show that the short-term consumption of diets composed entirely of animal or plant products alters microbial community structure and overwhelms inter-individual differences in microbial gene expression. The animal-based diet increased the abundance of bile-tolerant microorganisms (Alistipes, Bilophila and Bacteroides) and decreased the levels of Firmicutes that metabolize dietary plant polysaccharides (Roseburia, Eubacterium rectale and Ruminococcus bromii). Microbial activity mirrored differences between herbivorous and carnivorous mammals, reflecting trade-offs between carbohydrate and protein fermentation. Foodborne microbes from both diets transiently colonized the gut, including bacteria, fungi and even viruses. Finally, increases in the abundance and activity of Bilophila wadsworthia on the animal-based diet support a link between dietary fat, bile acids and the outgrowth of microorganisms capable of triggering inflammatory bowel disease. In concert, these results demonstrate that the gut microbiome can rapidly respond to altered diet, potentially facilitating the diversity of human dietary lifestyles.
Emerging next-generation sequencing technologies have revolutionized the collection of genomic data for applications in bioforensics, biosurveillance, and for use in clinical settings. However, to make the most of these new data, new methodology needs to be developed that can accommodate large volumes of genetic data in a computationally efficient manner. We present a statistical framework to analyze raw next-generation sequence reads from purified or mixed environmental or targeted infected tissue samples for rapid species identification and strain attribution against a robust database of known biological agents. Our method, Pathoscope, capitalizes on a Bayesian statistical framework that accommodates information on sequence quality, mapping quality and provides posterior probabilities of matches to a known database of target genomes. Importantly, our approach also incorporates the possibility that multiple species can be present in the sample and considers cases when the sample species/strain is not in the reference database. Furthermore, our approach can accurately discriminate between very closely related strains of the same species with very little coverage of the genome and without the need for multiple alignment steps, extensive homology searches, or genome assembly- which are time consuming and labor intensive steps. We demonstrate the utility of our approach on genomic data from purified and in silico 'environmental' samples from known bacterial agents impacting human health for accuracy assessment and comparison with other approaches.
A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera screening, standard alignment, and taxonomic classification
using multiple published taxonomies. It was found that there is incongruent taxonomic nomenclature among curators even at
the phylum level. Putative chimeras were identified in 3% of environmental sequences and in 0.2% of records derived from isolates.
Environmental sequences were classified into 100 phylum-level lineages in the Archaea and Bacteria.
Kwashiorkor, an enigmatic form of severe acute malnutrition, is the consequence of inadequate nutrient intake plus additional
environmental insults. To investigate the role of the gut microbiome, we studied 317 Malawian twin pairs during the first
3 years of life. During this time, half of the twin pairs remained well nourished, whereas 43% became discordant, and 7% manifested
concordance for acute malnutrition. Both children in twin pairs discordant for kwashiorkor were treated with a peanut-based,
ready-to-use therapeutic food (RUTF). Time-series metagenomic studies revealed that RUTF produced a transient maturation of
metabolic functions in kwashiorkor gut microbiomes that regressed when administration of RUTF was stopped. Previously frozen
fecal communities from several discordant pairs were each transplanted into gnotobiotic mice. The combination of Malawian
diet and kwashiorkor microbiome produced marked weight loss in recipient mice, accompanied by perturbations in amino acid,
carbohydrate, and intermediary metabolism that were only transiently ameliorated with RUTF. These findings implicate the gut
microbiome as a causal factor in kwashiorkor.
Microbial exposures and sex hormones exert potent effects on autoimmune diseases, many of which are more prevalent in women.
We demonstrate that early-life microbial exposures determine sex hormone levels and modify progression to autoimmunity in
the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D). Colonization by commensal microbes elevated serum testosterone
and protected NOD males from T1D. Transfer of gut microbiota from adult males to immature females altered the recipient's
microbiota, resulting in elevated testosterone and metabolomic changes, reduced islet inflammation and autoantibody production,
and robust T1D protection. These effects were dependent on androgen receptor activity. Thus, the commensal microbial community
alters sex hormone levels and regulates autoimmune disease fate in individuals with high genetic risk.
Symbiotic microorganisms that reside in the human intestine are adept at foraging glycans and polysaccharides, including those in dietary plants (starch, hemicellulose and pectin), animal-derived cartilage and tissue (glycosaminoglycans and N-linked glycans), and host mucus (O-linked glycans). Fluctuations in the abundance of dietary and endogenous glycans, combined with the immense chemical variation among these molecules, create a dynamic and heterogeneous environment in which gut microorganisms proliferate. In this Review, we describe how glycans shape the composition of the gut microbiota over various periods of time, the mechanisms by which individual microorganisms degrade these glycans, and potential opportunities to intentionally influence this ecosystem for better health and nutrition.
To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence, from faecal samples of 124 European individuals. The gene set, approximately 150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent) microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes. The genes are largely shared among individuals of the cohort. Over 99% of the genes are bacterial, indicating that the entire cohort harbours between 1,000 and 1,150 prevalent bacterial species and each individual at least 160 such species, which are also largely shared. We define and describe the minimal gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively.
Type 2 diabetes (T2D) is a result of complex gene-environment interactions, and several risk factors have been identified, including age, family history, diet, sedentary lifestyle and obesity. Statistical models that combine known risk factors for T2D can partly identify individuals at high risk of developing the disease. However, these studies have so far indicated that human genetics contributes little to the models, whereas socio-demographic and environmental factors have greater influence. Recent evidence suggests the importance of the gut microbiota as an environmental factor, and an altered gut microbiota has been linked to metabolic diseases including obesity, diabetes and cardiovascular disease. Here we use shotgun sequencing to characterize the faecal metagenome of 145 European women with normal, impaired or diabetic glucose control. We observe compositional and functional alterations in the metagenomes of women with T2D, and develop a mathematical model based on metagenomic profiles that identified T2D with high accuracy. We applied this model to women with impaired glucose tolerance, and show that it can identify women who have a diabetes-like metabolism. Furthermore, glucose control and medication were unlikely to have major confounding effects. We also applied our model to a recently described Chinese cohort and show that the discriminant metagenomic markers for T2D differ between the European and Chinese cohorts. Therefore, metagenomic predictive tools for T2D should be specific for the age and geographical location of the populations studied.
Dynamic protein binding to DNA elements regulates genome function and cell fate. Although methods for mapping in vivo protein-DNA interactions are becoming crucial for every aspect of genomic research, they are laborious and costly, thereby limiting progress. Here we present a protocol for mapping in vivo protein-DNA interactions using a high-throughput chromatin immunoprecipitation (HT-ChIP) approach. By using paramagnetic beads, we streamline the entire ChIP and indexed library construction process: sample transfer and loss is minimized and the need for manually labor-intensive procedures such as washes, gel extraction and DNA precipitation is eliminated. All of this allows for fully automated, cost effective and highly sensitive 96-well ChIP sequencing (ChIP-seq). Sample preparation takes 3 d from cultured cells to pooled libraries. Compared with previous methods, HT-ChIP is more suitable for large-scale in vivo studies, specifically those measuring the dynamics of a large number of different chromatin modifications/transcription factors or multiple perturbations.