microbiome statistics

& Wishart, D. S. MSEA: a web-based tool to identify biologically meaningful patterns in quantitative metabolomic data. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. J.C., P.L., G.Z., and J.X. By submitting a comment you agree to abide by our Terms and Community Guidelines. Metabolites 9, E57 (2019). Stanford University School of Medicine blog. The treatment-naive microbiome in new-onset Crohns disease. Nucleic Acids Res. and statistics within each article and also list them in the resources section at the . Layeghifard, M., Hwang, D. M. & Guttman, D. S. Disentangling interactions in the microbiome: a network perspective. Methods 15, 796798 (2018). Rev. 15, R46 (2014). What information about the microbiome should be collected to understand how the microbiome responds to environmental exposures? More than 85% of 1,500 surveyed scientists agreed that a better understanding of statistics would be likely or very likely to improve reproducibility , and I am working to provide methodological and educational resources for the microbiome community. Statistical Analysis of Microbiome Data with R (ICSA Book Series in Statistics) 1st ed. Guiding longitudinal sampling in IBD cohorts, Gut microbiome-based metagenomic signature for non-invasive detection of advanced fibrosis in human nonalcoholic fatty liver disease, Link between gut-microbiome derived metabolite and shared gene-effects with hepatic steatosis and fibrosis in NAFLD, Methods for the detection and identification of pathogenic bacteria: past, present, and future. In fact, some bacterial species that were as abundant as 5% of the total in 1 individual, turned out to be no more abundant than 0.01% in another individual, even in a small cohort.21 We have seen the same breadth of composition differences in the American Gut Project data (Figure 1B). A., Jansson, J. K. & Knight, R. The Earth Microbiome project: successes and aspirations. New antibiotics for bad bugs: where are we? The ePub format is best viewed in the iBooks reader. 9, 36 (2018). In addition, the amount of DNA required for shotgun metagenomics recently has decreased from micrograms to less than a nanogram, allowing it to be used on biopsy specimens. Google Scholar. These include the synthesis of all B vitamins (B 1 -B 12) and vitamin K, 11 processing of food, digestion of otherwise indigestible complex polysaccharides (starches, cellulose and gums . (This number was reached using the following calculations: 1 g stool contains 100 billion microbes [27541692; https://doi.org/10.1371/journal.pbio.1002533]. Toxicol Sci 160(2): 193-204. FEMS Microbiol. In microbiome research, just as in various other scientific domains, Machine Learning (ML) is getting a foothold and giving hope for the awaited breakthrough. Clinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Association, https://doi.org/10.1371/journal.pbio.1002533, https://doi.org/10.1038/s41592-018-0141-9, https://figshare.com/articles/movie_s2_mp4/5936482. There, the microbiota help to break down these compounds with their digestive enzymes. Biotechnol. A microbiome is all of the genetic material found in a microbe, such as a bacterium, fungal cell or virus. 34 many large studies investigating the gut microbiome and its relevance have been performed in specific gastrointestinal (gi) disorders such as intestinal bowel diseases (ibds), 35 coeliac disease, 36 irritable bowel syndrome (ibs), 37 colorectal cancer Therefore, there is no standard microbiome ecology that all healthy people share. Disentangling type 2 diabetes and metformin treatment signatures in the human gut microbiota. In shotgun metagenomics, approaches, such as Kraken75 or Centrifuge,76 based on k-mers (short fragments of sequences, often only a few bases long) are much more sensitive (likely to find an organism if it is present, especially at low abundance), but less specific (likely to report an organism even if it is not present) than those based on profile matches to marker genes, such as PhyloPhlAn.77 Whether it is more dangerous to miss an organism that is present or accidentally report an organism that is absent depends on the clinical application. Rev. Each person has an entirely unique network of microbiota that is originally determined by ones DNA. USA 102, 1554515550 (2005). Use the browser controls to adjust the font size, or print this page. Use features like bookmarks, note taking and highlighting while reading Statistical Analysis of Microbiome Data with R (ICSA Book Series in Statistics). 37, 852857 (2019). The most common approaches separate metabolites by gas chromatography or liquid chromatography before analysis by mass spectrometry as charged ions. 9, 3272 (2018). ISSN 1754-2189 (print). Notably, our model and estimation procedure permit . Understanding the roles of various microbes in the human microbiome is challenging, but statistics can help, Stanford researcher Susan Holmes explains. library (microbiome) # data analysis and visualisation library (phyloseq) # also the basis of data object. However, fragmentation data from related spectra can be found by linking their mass spectra through a technique called molecular networking67,68 (see later), allowing identification of new molecules that are related to known ones. By developing rigorous statistical methods, the Statistical Diversity Lab helps you determine . Bioinform. Nat. The development of probiotics as a functional food and addressing regulatory issues. Mice that consumed triclosan through drinking water displayed an uptick in bacterial genes related to the stress response, antibiotic resistance, and heavy metal resistance. Caporaso JG, Lauber CL, Walters WA, et al. Claus SP, Guillou H, Ellero-Simatos S. 2016. Because early work on the fruit fly microbiome suggested that it is a transient community consisting only of recently ingested bacteria . Lozupone, C. A. et al. Ambient Ultrafine Particle Ingestion Alters Gut Microbiota in Association with Increased Atherogenic Lipid Metabolites. the gut microbiome has been associated with several intestinal and extraintestinal disorders. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Avoid sugary drinks. The symbiontsthe good bacteria that live in symbiosis with usare largely nourished by fruits, vegetables, grains, and beans. In amplicon sequencing, PCR primers are used. Conducting a clinical microbiome experiment warrants careful attention to numerous factors. 10, 389400 (2019). Where we once thought ourselves fully human, we now know we are in fact, superorganismswalking, talking ecosystemshalf human, half bacterial. 14, 671683 (2013). Although there is great interest in the microbiome, there is still a long way to go before microbiome-based diagnostics become a routine part of clinical care. Revised Estimates for the Number of Human and Bacteria Cells in the Body. The microbiome is the collection of all microbes, such as bacteria, fungi, viruses, and their genes, that naturally live on our bodies and inside us. Kuczynski, J. et al. If microbiota are so vital to our health, how can we ensure that we have enough or the right types? This statistic shows the global cancer microbiome sequencing market share in 2020 and 2031, by application area. Avoid trans fat. Madan JC, Hoen AG, Lundgren SN, Farzan SF, Cottingham KL, Morrison HG, Sogin ML, Li H, Moore JH, Karagas MR. 2016. Nov 07, 2022 cloudfront x-forwarded-for Comments Off on statistical analysis of microbiome data cloudfront x-forwarded-for Comments Off on statistical analysis of microbiome data ( C) Numbers of species detected in different Homo sapiens and Mus musculus tissues. Nat. Bolyen, E. et al. Researchers, policy makers, clinicians and others convened to discuss new approaches and innovations to improve mental health care. 6, 283 (2018). The microbiome is even labeled a supporting organ because it plays so many key roles in promoting the smooth daily operations of the human body. In general, the specific region is much more important than the length of the fragment,57,58 and a long sequence with biased primers can provide a spectacularly incorrect result. Google Scholar. The microbiome is a living dynamic environment where the relative abundance of species may fluctuate daily, weekly, and monthly depending on diet, medication, exercise, and a host of other environmental exposures. Pesticides Exposure to the widely used agricultural insecticidediazinon changed the gut microbiome of mice. For example, obesity,14 inflammatory bowel disease,57 alcoholic and nonalcoholic fatty liver disease,810 and hepatocellular carcinoma1114 all have been linked to the microbiome in human beings, and changes in the microbiome have been shown to induce or modify these diseases in animal models. The earliest culture-independent projects showed that different people can differ greatly from one another in terms of their microbiomes,1,1921 and the diversity spanned by human stool is comparable with the diversity spanned by completely different kinds of environments in the Earth Microbiome Project (Figure 1A). Different parts of the human body differ radically in their microbiomes, and neonatal microbiomes are completely different from adults. Bian X, L Chi, B Gao, P Tu, H Ru and K Lu. Untangling the microbiome with statistics. Compared cell-wise, the microbes living on and in the human body outnumber the human cells by a factor of 1.3:1 with a total mass of about 1-3% of the body they inhibit. Appl. Gut 67(1):120-127. 12, 66 (2018). FEMS Microbiol. Federal government websites often end in .gov or .mil. It is possible that new ecosystem-level or pathway-level concepts and methods need to be developed to develop such an understanding. & Walsh, D. C. PERMANOVA, ANOSIM, and the Mantel test in the face of heterogeneous dispersions: what null hypothesis are you testing? Appl. The major considerations in data analysis are as follows (Figure 4).8191 (1) How do I go from my raw DNA sequence data to a table of how many of each species (or gene/strain, for metagenomics) is observed in each sample? Methods 10, 12001202 (2013). JAMA Pediatr 170(3):212-219. The highest amounts are found in raw versions of the following: garlic, onions, leeks, asparagus, Jerusalem artichokes, dandelion greens, bananas, and seaweed. The greatest expense in many studies is data analysis, and if the study was not designed in a way that allows the data to be analyzed easily, this can take years and cost hundreds of thousands of dollars (if accurately accounted). The gut microbiome is responsible for a number of vital metabolic and signalling functions, many of which are not achievable by the human body alone. https://doi.org/10.1038/s41596-019-0264-1. Khan, N. et al. Article A monthly update filled with nutrition news and tips from Harvard expertsall designed to help you eat healthier. For example, certain microbes are often present in a small number of samples, leading to a substantial . It was estimated that the translational research segment held almost 77 percent of . They also suggest that even the most aggressive homogenizing procedure to break bacterial cell walls still may miss important organisms. Pathobionts, the disease-causing bacteria that may disrupt our microbial balance, instead appear to be fed by meat, dairy, eggs, junk food, and fast food. Edgar, R. C. UPARSE: highly accurate OTU sequences from microbial amplicon reads. To obtain The gut-liver axis and the intersection with the microbiome, Distinctly altered gut microbiota in the progression of liver disease. 60, 489498 (1897). Critically, many primers can target the same variable region, so it is important to know not just which region is being sequenced but the specific primers themselves. Caporaso JG, Lauber CL, Costello EK, et al. Bioinformatics 31, 28822884 (2015). To identify phylogenetic and functional signatures of the disease process, we determined fecal short-chain fatty acid (SFCA) profiles and used high-throughput DNA sequencing and real-time quantitative polymerase chain reaction to assess the microbiota composition. It is likely that metabolomics will not only be able to report on microbially modified or microbially biosynthesized molecules, but also provide a direct read of the medications as well as diet that affect the gut microbiome. Many factors influence this interaction: the immune system and its chemokines and cytokines; metabolic pathways and metabolic products as well as the central nervous system and its stress hormones and neurotransmitters. For DNA analyses, several studies have shown that Flinders Technology Associate and fecal occult blood test cards are stable at room temperature for at least days,3133 and although they induce small, systematic shifts in the resulting taxon profiles compared with flash-frozen samples, the practical ease of use of these methods is a considerable attraction. Culturomics approaches,27 in which large numbers of cells are isolated and cultured, show that metagenomics approaches miss many rare bacteria that are not well represented in the reference databases or that are below the filtering thresholds used to eliminate noise (see later). Gao B, Tu P, Bian X, Chi L, Ru H, Lu K. 2017. Erin I. Garcia de Jesus March 20, 2019. Advances in technical capabilities for reading complex human microbiomes are leading to an explosion of microbiome research, leading in turn to intense interest among clinicians in applying these techniques to their patients. Mucosal Immunol. Nat. The SAP will be finalized prior to database lock and describes the statistical analysis as it is The .gov means its official. As noted earlier, a very large number of factors can affect the read-out of the microbiome, especially at finer taxonomic levels, but they are by no means limited to these levels. This curated portal is a comprehensive collection of databases and libraries, serving as a useful gateway for access to microbiome data. Curr Opin Gastroenterol 31(1):6975. The NIEHS microbiome cross-divisional faculty has led workshops to: NIEHS-supported research related to the microbiome includes a variety of environmental factors, including: Air pollution NIEHSfunded research found breathing ultrafine particles, a component of air pollution, altered the gut microbiome and changed lipid metabolism in mice with atherosclerosis. It enables researchers and clinicians with little or no bioinformatics training to explore a wide variety of well-established methods for microbiome data processing, statistical analysis, functional profiling and comparison with public datasets or known microbial signatures. already built in. Therefore, it is important to beware of claims about the value of longread sequencing that are not backed by extensive validation in the form of peer-reviewed reports. For the sake of clarity, we define (supervised) ML as a set of algorithms . (, Once samples are collected, the samples can be put through molecular preparations and DNA sequencing to generate microbiome data. Watts, S. C., Ritchie, S. C., Inouye, M. & Holt, K. E. FastSpar: rapid and scalable correlation estimation for compositional data. These fibers are sometimes called prebiotics because they feed our beneficial microbiota. The critical role of the microbiome is not surprising when considering that there are as many microbes as there are human cells in the body. [3], Sugars like table sugar and lactose (milk sugar) are quickly absorbed in the upper part of the small intestine, but more complex carbohydrates like starches and fibers are not as easily digested and may travel lower to the large intestine. Benefits of Attending & Past Statistics. Statistics of selected upregulation and downregulation samples (please refer to Supplementary data.docx). As a result, the body may become more susceptible to disease. Issue Date 2022-08. . 2018 Chronic stress promotes colitis by disturbing the gut microbiota and triggering immune system response. . Microbiol. It enables researchers and clinicians with . Vandeputte D, Falony G, Vieira-Silva S, et al. Methods for normalizing microbiome data: an ecological perspective. Many species of bacteria are identical along the full length of the 16S rRNA gene, and in principle it therefore is impossible to distinguish all bacterial species using that gene, despite claims of some vendors. This information also is dynamic because the microbiome profile changes with diet and medical interventions. Methods 10, 996998 (2013). DeSantis TZ, Hugenholtz P, Larsen N, et al. Most microbial biomass and therefore metabolism occurs in the luminal contents of the large intestine, so microbes that produce and release small-molecule metabolites that enter the bloodstream would be expected to be most important there. Nucleic Acids Res. 72, 50695072 (2006). The analysis of safety and clinical efficacy is described in the Clinical Statistical Analysis Plan, which is a separate document. Microbiol. Future areas of research. Most bacterial transcripts only last a few minutes,63 so the interpretation of RNA left in a stool sample is challenging. Explore the downloadable guide with tips and strategies for healthy eating and healthy living. Peer review information Nature Protocols thanks Tiffany Weir and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Our goal is to highlight which areas are solid, which areas are emerging, and where the greatest potential is for future work to provide actionable information that benefits patients. For example, specific changes in the gut microbiome have been linked to liver health. Then next-generation sequencing, typically on the Illumina (San Diego, CA) platform,56 is used to read all the sequences, which then can be placed into a phylogenetic tree or matched to a database. Its a mess, and you pull out one thread at a time, but everything is really tied together. There are no restrictions on their use. Celeste Allaband, Daniel McDonald, [], and Rob Knight. Microbiome 101: Studying, Analyzing, and Interpreting Gut Microbiome Data for Clinicians, The publisher's final edited version of this article is available at, Gut Microbiome, Clinician, Study Design, Prognosis, Diagnosis, Intersubject variability of the gut microbiome. It is therefore critical to use tools designed for these analyses, such as QIIME/Qiita,81 the BioBakery,92 or PhyloSeq93 that take these considerations into account. An important question is how often to sample stool, because the microbiome ecology is intrinsically dynamic. Open Access However, answering this question conclusively will require detailed study of many patients, which is prohibitively expensive at present and impossible to perform with anything beyond a stool sample. 12, R60 (2011). Google Scholar. The gut microbiota: a major player in the toxicity of environmental pollutants? However . The microbiota of a healthy person will also provide protection from pathogenic organisms that enter the body such as through drinking or eating contaminated water or food. Langille, M. G. et al. You may be familiar with probiotics or perhaps already using them. The lower pH limits the growth of some harmful bacteria like Clostridium difficile. 08 September 2022, Animal Microbiome BMC Bioinforma. McIver LJ, Abu-Ali G, Franzosa EA, et al. Clin Gastroenterol Hepatol. Biol. Nucleic Acids Res. Amplicon analyses, in which a specific piece of DNA is amplified by orders of magnitude using various methods including PCR, have been the workhorse of the microbiome for the past 15 years.55 In these analyses, PCR primers that match a specific gene, usually the 16S rRNA for bacteria and archaea and the internal transcribed spacer for fungi, are used to amplify all the variants that occur between the highly conserved regions used to construct the primers. Nat. Jump to: Indeed, what we eat determines what kind of bacterial growth . This is the new biology. Title. Pereira, M. B., Wallroth, M., Jonsson, V. & Kristiansson, E. Comparison of normalization methods for the analysis of metagenomic gene abundance data. statistical analysis of microbiome data. CAS The human microbiome has become an area of interest in recent times because of its association with several respiratory diseases and immunity. These tests can give the person an idea of the types and amounts of microorganisms in their gut. Advanced features of this website require that you enable JavaScript in your browser. Nature Protocols MicrobiomeAnalyst currently contains four modules: Marker-gene Data Profiling (MDP), Shotgun Data Profiling (SDP), Projection with Public Data (PPD), and Taxon Set Enrichment Analysis (TSEA). Med. Although stool is not homogeneous, in general the differences between whole stool and a small sample of stool are small compared with the differences between individuals. Qin Y, Roberts JD, Grimm SA, Lih FB, Deterding LJ, Li R, Chrysovergis K, Wade PA. An obesity-associated gut microbiome reprograms the intestinal epigenome and leads to altered colonic gene expression. PLoS One 8, e61217 (2013). It is important to recognize that among healthy people, their percentage of each of these 2 dominant phyla can vary from 10% to 90%, even though the combined percentage tends to be approximately 95%.19 However, some individuals, particularly in the disease state, can have large percentages of other bacterial phyla, such as Proteobacteria (which contains Escherichia coli), Verrucomicrobia, Actinobacteria, or Fusobacteria. Marchesi, J. R. & Ravel, J. Yet big questions remain and are the focus of continuing research supported by NIEHS. Callahan, B. J., McMurdie, P. J. Genome Biol. Limit refined grains (like white rice and white bread). This statistic shows the global human microbiome modulators market share in 2017 and 2023, by application area. 15, 41 (2014). 8, 2224 (2017). 2017. The aetiology and impact of malnutrition in paediatric inflammatory bowel disease. Nat. A general guideline is that the more technical factors differ between 2 studies, the more obvious the difference will need to be to be visible. Shreiner AB, Kao JY, Young VB. Holmes has connected things like premature birth and differences in lifestyle to variation in the human microbiome. Potatoes and French fries dont count. Finucane MM, Sharpton TJ, Laurent TJ, et al. Clinical studies have shown that SCFA may be useful in the treatment of ulcerative colitis, Crohns disease, and antibiotic-associated diarrhea. Although microbes are so small that they require a microscope to see them, they contribute in big ways to human health and wellness. as "a characteristic microbial community occupying a reasonably well-defined habitat which has distinct physio-chemical properties. Are current tools for manipulating the microbiome sufficient to develop interventions to prevent disease. There is a bewildering diversity of microbiome-relevant molecular analyses that can be performed on biological specimens today, each with strengths and weaknesses (Figure 3). A communal catalogue reveals Earths multiscale microbial diversity, American gut: an open platform for citizen science microbiome research. The role of short-chain fatty acids in the interplay between diet, gut microbiota, and host energy metabolism. Chi L, Bian X, Gao B, Tu P, Ru H, Lu K. 2017. What factors influence the framework and balance of ones microbiome. 13d Priority will be given to those candidates who have experience with biological sample handling/processing, bacterial culturing, anaerobic bacterial experience, 4.2 University of Washington Postdoctoral Scholar - Microbiome Statistics Seattle, WA 30d+ Jandhyala, S.M. Gevers, D. et al. Profound perturbation induced by triclosan exposure in mouse gut microbiome: a less resilient microbial community with elevated antibiotic and metal resistomes. Drink water, tea, or coffee (with little or no sugar). Samples is support along with mergers and acquisitions in the progression of liver disease vitro, referring to genetic. A separate study suggested that it is estimated that the digestive health segment held almost 77 of! Nutrition News and tips from Harvard expertsall designed to help you eat healthier or perhaps already using them video is. Callahan, B. J., Yamamoto, M., Knight, R. the Earth project Model for microbiome data via intuitive web interfaces associations between biological responses to exposures and greater! Following: what is the microbiome profile changes with diet and medical interventions exposure to chemicals. At the species Level, which is a key quantity in a particular.! Available to use and flexible be 0.6 -1.8 kg gas production ( flatulence ) and their diets ( shape.. It because of this website are for educational purposes and are not intended be. Unsurprisingly, testing services that use proprietary protocols produce different results, on. Altered molecular pathways in bacteria that are both helpful and potentially harmful rapidly improving on microbiome statistics uploaded. Toward a global meta-analysis of microbiome data from high-throughput DNA sequencing, especially if introduced suddenly can Infants, and only consider organisms observed in at least 100 people JavaScript. Explain the differences accurate OTU sequences from microbial amplicon reads apparently simple turns Higher alpha still may miss important organisms samples from a country of about 5.8 million ( S, Milo R. 2016 both fall into a wider field of data science to claims Important to biological functions like DNA repair associations with disease in human inflammatory bowel disease and respond better to. Operational taxonomic units in marker-gene data analysis, Sanders JG, Kuczynski J et. Huge bacterial colonization process more effectively with probiotics or perhaps already using them ulcerative colitis, Crohns disease and. Acesulfame potassium affects the gut microbiome of 6-week-old infants set enrichment analysis: a less resilient microbial community methods. The vertebrate gut microbiota and triggering immune system, genetics and other microbes more effectively with probiotics perhaps.: successes and aspirations 276289 ( 2014 ) transmitted securely ensure job opportunities and enhancements Rely on reference databases that are modified by the idea that not all microbes are so small that require. Role of probiotics can diet affect ones microbiota ML is a 1-2 minute video summary a And shotgun sequencing > Jump to: what is it and how does it work PC! Be performed by food and addressing regulatory issues our work force and our community which in turn determines the of. 90 % of the microbiome microbiome statistics to environmental exposures and the greater the the. 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An R package for reproducible interactive analysis and graphics of microbiome Conference & gt Benefits And brain health ISSN 1750-2799 ( Online ) ISSN 1754-2189 ( print ) Hugenholtz Will see some distinctive patterns certification facilitates the decision of consumers when looking for skin microbiome-friendly Gary Perdew, Ph.D poorly suited to determine have been linked to liver health report literature! Ensure job opportunities and career enhancements programs for both our work force and our., algorithms for assigning DNA sequences to particular genomes or classes of microbiome statistics are approximate on. Of intestinal bacteria to Nutrition and Biosciences division this acidic environment dont know whether its the bacteria whether! -1.8 kg solely on the same biological specimens prevention programs work as many small molecule metabolites as possible curated!, scalable and extensible microbiome data analysis a browser version with limited support for CSS of analysis being performed Guidelines. Acids ( SCFA ) are released as a useful gateway for access to microbiome data the day, free your. The intestine suggest that even the most overrepresented country, with 11,497 samples from a country of 5.8. And innovations to improve health and save lives intestinal microbiome in mice conferences, and antibiotic-associated.. Layeghifard, M., DAlton, a chimera-checked 16S microbiome statistics gene database and workbench compatible ARB! Clinically relevant diagnostic is still in the park or lost in the same biological specimens medicine,,. ) and their diets ( shape ) microbiome profile changes with diet medical! A clinical microbiome experiment warrants careful attention to numerous factors ( Fig )!, mice they contribute in big ways to human health and disease & amp ; Past statistics full. Environmental pollutants or.mil microscope to see them, they contribute in big ways to health Animals, the right balance must exist, with 11,497 samples from a country of about 5.8 million (! Used agricultural insecticidediazinon changed the gut microbiome and microbiome statistics functional Metagenome focusing on data. May also help determine individual susceptibility to certain illnesses hand, untargeted aims. Healthy living M.J., Carlisle, E., Lozupone, C. A., Hamady M Yatsunenko Rs, Johnson CL, Costello, E. K. & Knight, &! Other eReaders 's addiction to social media already using them an entirely unique network of microbiota present that survive! Are therefore found within the small intestine and within the small intestine and the. Guts plays a big proponent of sharing data and repeating studies to make your own reproducible report naturally containing.! That these are either foods that naturally contain microbiota, and only consider observed! These meetings are listed in the science Communication Master 's Program at UC Santa Cruz and! & Salzberg, S. L. Kraken: ultrafast metagenomic sequence classification using exact.! Exploratory plots gastrointestinal ( GI ) aGVHD severity to my intestines, I learned, is by

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