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Title: The Salivary Microbiome is Altered in Children with Eosinophilic Esophagitis and it Correlates with Disease Activity.Short title: Salivary Microbiome in Eosinophilic EsophagitisAuthors:Girish Hiremath, MD MPHDivision of Pediatric Gastroenterology, Hepatology and Nutrition2200 Children’s way, 10th FloorVanderbilt University Medical Center, Nashville TN 37232Meghan H. Shilts, MHS MSDivision of Infectious DiseasesVanderbilt University Medical Center?1211, 21st Avenue SouthS2108 Medical Center North, Nashville, TN 37232?Helen H. Boone, BASDivision of Infectious DiseasesVanderbilt University Medical Center?1211, 21st Avenue SouthS2108 Medical Center North, Nashville, TN 37232? Hernan Correa, MDDivision of Pediatric Pathology2200 Children’s way, 11th FloorVanderbilt University Medical Center, Nashville, TN 37232Sari Acra, MD MPHDivision of Pediatric Gastroenterology, Hepatology and Nutrition2200 Children’s way, 10th FloorVanderbilt University Medical Center, Nashville TN 37232Andrey Tovchigrechko, PhDResearch BioinformaticsMedImmune1 MedImmune Way, Gaithersburg, MD 20878 Seesandra V. Rajagopala, PhDDivision of Infectious DiseasesVanderbilt University Medical Center?1211, 21st Avenue SouthS2108 Medical Center North, Nashville, TN 37232? Suman R. Das, PhDDivision of Infectious DiseasesVanderbilt University Medical Center?1211, 21st Avenue SouthS2108 Medical Center North, Nashville, TN 37232?Corresponding authors:Suman R. DasEmail: suman.r.das@vanderbilt.eduPhone: 615-322-0322Seesandra V. RajagopalaE-Mail: s.v.rajagopala@vanderbilt.eduPhone: 615-322-2419Address:Division of Infectious DiseasesVanderbilt University Medical Center?1211, 21st Avenue SouthS2108 Medical Center NorthNashville, TN 37232?Word count:Manuscript: 3361Tables: 1Figures: 4Supplementary material: 1SUPPLEMENTARY MATERIALS AND METHODS Data processing and statistical analysisReads were processed by following the mothur MiSeq SOP (wiki/MiSeq_SOP) as of August 4, 2017.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Lb3ppY2g8L0F1dGhvcj48WWVhcj4yMDEzPC9ZZWFyPjxS
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ADDIN EN.CITE.DATA 1 Reads were aligned against the SILVA database release 128 ADDIN EN.CITE <EndNote><Cite><Author>Pruesse</Author><Year>2007</Year><RecNum>54</RecNum><DisplayText><style face="superscript">2</style></DisplayText><record><rec-number>54</rec-number><foreign-keys><key app="EN" db-id="zas9txe9ketev1evxvwpdtdqsdd0rev95z2e" timestamp="1472226895" guid="b31022e1-b529-45be-b352-eaadf68596c7">54</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Pruesse, Elmar</author><author>Quast, Christian</author><author>Knittel, Katrin</author><author>Fuchs, Bernhard M.</author><author>Ludwig, Wolfgang</author><author>Peplies, J?rg</author><author>Gl?ckner, Frank Oliver</author></authors></contributors><titles><title>SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB</title><secondary-title>Nucleic acids research</secondary-title></titles><periodical><full-title>Nucleic Acids Res</full-title><abbr-1>Nucleic acids research</abbr-1></periodical><pages>7188-96</pages><volume>35</volume><number>21</number><keywords><keyword>Base Sequence</keyword><keyword>Databases, Nucleic Acid</keyword><keyword>Databases, Nucleic Acid: standards</keyword><keyword>Genes, rRNA</keyword><keyword>Internet</keyword><keyword>Phylogeny</keyword><keyword>Quality Control</keyword><keyword>RNA, Ribosomal</keyword><keyword>RNA, Ribosomal: genetics</keyword><keyword>Sequence Alignment</keyword><keyword>Sequence Analysis, RNA</keyword><keyword>Software</keyword></keywords><dates><year>2007</year></dates><urls><related-urls><url> and taxonomy was assigned using the RDP database version 11.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Db2xlPC9BdXRob3I+PFllYXI+MjAwOTwvWWVhcj48UmVj
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ADDIN EN.CITE.DATA 3 Operational taxonomic units (OTUs) were clustered at 97% similarity.Descriptive statistics were used to characterize the cohort. Microbiome analysis was performed in R. The majority of the analyses were done using the open source package MGSAT, which wraps several R packages in order to perform -omics analyses []. Figures were generated with the R package ggplot2. ADDIN EN.CITE <EndNote><Cite><Author>Wickham</Author><Year>2009</Year><RecNum>128</RecNum><DisplayText><style face="superscript">4</style></DisplayText><record><rec-number>128</rec-number><foreign-keys><key app="EN" db-id="zas9txe9ketev1evxvwpdtdqsdd0rev95z2e" timestamp="1472226970" guid="04fab66e-f03f-48f3-8e9b-7dabd8ff13b0">128</key></foreign-keys><ref-type name="Book">6</ref-type><contributors><authors><author>Wickham, Hadley</author></authors></contributors><titles><title>ggplot2: elegant graphics for data analysis</title></titles><dates><year>2009</year></dates><pub-location>New York</pub-location><publisher>Springer</publisher><urls></urls></record></Cite></EndNote>4 Associations were considered significant if the p- or q-value (as appropriate) was < 0.05.Significant associations between clinical, endoscopic, or histologic metadata and bacterial taxa at the OTU and genus levels were assessed using the R package DESeq2. ADDIN EN.CITE <EndNote><Cite><Author>Love</Author><Year>2014</Year><RecNum>13</RecNum><DisplayText><style face="superscript">5</style></DisplayText><record><rec-number>13</rec-number><foreign-keys><key app="EN" db-id="tpv9a29wvewt07ex5aepvxx1a59r9e9da2sw" timestamp="1536725303">13</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Love, M. I.</author><author>Huber, W.</author><author>Anders, S.</author></authors></contributors><titles><title>Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2</title><secondary-title>Genome Biol</secondary-title></titles><periodical><full-title>Genome Biol</full-title></periodical><pages>550</pages><volume>15</volume><number>12</number><keywords><keyword>Algorithms</keyword><keyword>Computational Biology/*methods</keyword><keyword>High-Throughput Nucleotide Sequencing</keyword><keyword>Models, Genetic</keyword><keyword>RNA/*analysis</keyword><keyword>Sequence Analysis, RNA</keyword><keyword>*Software</keyword></keywords><dates><year>2014</year></dates><isbn>1474-760X (Electronic)
1474-7596 (Linking)</isbn><accession-num>25516281</accession-num><urls><related-urls><url> To reduce the penalty associated with multiple testing and to remove likely non-informative features, prior to DESeq2 analysis, taxa with an average relative abundance < 0.0005 were aggregated into a group called “other” and this group was included during testing but was otherwise ignored. For the DESeq2 analyses, we built both unadjusted and adjusted models. Our adjusted models included covariates expected a priori to influence the salivary microbiome: age, gender, ethnicity, and exposure to medications. Reported q-values are the result of a Wald test with the Benjamini-Hochberg correction ADDIN EN.CITE <EndNote><Cite><Author>Benjamini</Author><Year>1995</Year><RecNum>121</RecNum><DisplayText><style face="superscript">6</style></DisplayText><record><rec-number>121</rec-number><foreign-keys><key app="EN" db-id="zas9txe9ketev1evxvwpdtdqsdd0rev95z2e" timestamp="1472226970" guid="7c57b4fb-6ac1-47c1-b2c5-9090e07babb2">121</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Benjamini, Yoav</author><author>Hochberg, Yosef</author></authors></contributors><titles><title>Controlling the false discovery rate: a practical and powerful approach to multiple testing</title><secondary-title>J R Stat Soc Series B Stat Methodol</secondary-title></titles><periodical><full-title>J R Stat Soc Series B Stat Methodol</full-title></periodical><pages>289-300</pages><volume>57</volume><number>1</number><dates><year>1995</year></dates><urls></urls><electronic-resource-num>10.2307/2346101</electronic-resource-num></record></Cite></EndNote>6 applied to adjust for multiple comparisons. Richness and alpha- and beta-diversity metrics were calculated with the R package vegan ADDIN EN.CITE <EndNote><Cite><Author>Oksanen</Author><Year>2014</Year><RecNum>77</RecNum><DisplayText><style face="superscript">7</style></DisplayText><record><rec-number>77</rec-number><foreign-keys><key app="EN" db-id="zas9txe9ketev1evxvwpdtdqsdd0rev95z2e" timestamp="1472226944" guid="244d0a75-d5a5-4512-ae66-d8e612106d65">77</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Oksanen, Jari</author><author>Blanchet, F. Guillaume</author><author>Kindt, Roeland</author><author>Legendre, Pierre</author><author>Minchin, Peter R.</author><author>O'Hara, R. B.</author><author>Simpson, Gavin L.</author><author>Solymos, Peter</author><author>Stevens, M. Henry H.</author><author>Wagner, Helene</author></authors></contributors><titles><title>vegan: Community Ecology Package. R package version 2.0-10.</title></titles><dates><year>2014</year></dates><urls><related-urls><url> at the OTU level; all OTUs regardless of abundance were included. To control for differences in sequencing depth per sample, samples were randomly rarefied to the minimum sample read count and then each richness, alpha- or beta-diversity index was calculated. For each index, this process was repeated 400 times and results were averaged. Beta diversity was assessed with the Bray-Curtis dissimilarity index and the PermANOVA test as implemented in Adonis ADDIN EN.CITE <EndNote><Cite><Author>Anderson</Author><Year>2001</Year><RecNum>140</RecNum><DisplayText><style face="superscript">8</style></DisplayText><record><rec-number>140</rec-number><foreign-keys><key app="EN" db-id="zas9txe9ketev1evxvwpdtdqsdd0rev95z2e" timestamp="1472226975" guid="775e59a9-c51a-4db0-bb1d-d8e584f7b8b4">140</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Anderson, Marti J</author></authors></contributors><titles><title>A new method for non-parametric multivariate analysis of variance</title><secondary-title>Austral Ecol</secondary-title></titles><periodical><full-title>Austral Ecol</full-title></periodical><pages>32-46</pages><volume>26</volume><number>1</number><keywords><keyword>ANOVA</keyword><keyword>distance measure</keyword><keyword>experimental design</keyword><keyword>linear model</keyword><keyword>multifactorial</keyword><keyword>multivariate dissimilarity</keyword><keyword>partitioning</keyword><keyword>permutation tests</keyword><keyword>statistics</keyword></keywords><dates><year>2001</year></dates><publisher>Blackwell Science Pty</publisher><isbn>1442-9993</isbn><urls><related-urls><url> was used to test for significant differences between overall microbial composition and metadata groupings. Richness was assessed by calculating the abundance-based S. chao ADDIN EN.CITE <EndNote><Cite><Author>Chao</Author><Year>1987</Year><RecNum>4</RecNum><DisplayText><style face="superscript">9</style></DisplayText><record><rec-number>4</rec-number><foreign-keys><key app="EN" db-id="zas9txe9ketev1evxvwpdtdqsdd0rev95z2e" timestamp="1472226795" guid="76dd253b-0b9b-44aa-b6ff-361cdf1b3090">4</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Chao, A.</author></authors></contributors><titles><title>Estimating the population size for capture-recapture data with unequal catchability</title><secondary-title>Biometrics</secondary-title></titles><periodical><full-title>Biometrics</full-title></periodical><pages>783-91</pages><volume>43</volume><number>4</number><keywords><keyword>Animals</keyword><keyword>Biometry</keyword><keyword>Ecology</keyword><keyword>Models, Biological</keyword><keyword>Population</keyword><keyword>Rabbits</keyword></keywords><dates><year>1987</year></dates><urls><related-urls><url> index and by estimating the number of OTUs in each sample (hereafter referred to as S.obs). Alpha diversity was assessed using Hill numbers N1 and N2, which are, respectively, the exponential of the Shannon index and the inverted Simpson index. ADDIN EN.CITE <EndNote><Cite><Author>Hill</Author><Year>1973</Year><RecNum>127</RecNum><DisplayText><style face="superscript">10</style></DisplayText><record><rec-number>127</rec-number><foreign-keys><key app="EN" db-id="zas9txe9ketev1evxvwpdtdqsdd0rev95z2e" timestamp="1472226970" guid="6c560228-6911-482c-9e1f-b395fe004a10">127</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Hill, M O</author></authors></contributors><titles><title>Diversity and evenness: a unifying notation and its consequences</title><secondary-title>Ecology</secondary-title></titles><periodical><full-title>Ecology</full-title></periodical><pages>427-432</pages><volume>54</volume><number>2</number><dates><year>1973</year></dates><urls></urls><electronic-resource-num>10.2307/1934352</electronic-resource-num></record></Cite></EndNote>10 Generalized linear models were fit to test for significant associations between metadata categories and richness/alpha-diversity indices. REFERENCES ADDIN EN.REFLIST 1Kozich, J. J., Westcott, S. L., Baxter, N. T., Highlander, S. K. & Schloss, P. D. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Applied and environmental microbiology 79, 5112-5120, doi:10.1128/aem.01043-13 (2013).2Pruesse, E. et al. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic acids research 35, 7188-7196, doi:10.1093/nar/gkm864 (2007).3Cole, J. R. et al. The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic acids research 37, D141-145, doi:10.1093/nar/gkn879 (2009).4Wickham, H. ggplot2: elegant graphics for data analysis. (Springer, 2009).5Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15, 550, doi:10.1186/s13059-014-0550-8 (2014).6Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Series B Stat Methodol 57, 289-300, doi:10.2307/2346101 (1995).7Oksanen, J. et al. vegan: Community Ecology Package. R package version 2.0-10. (2014).8Anderson, M. J. A new method for non-parametric multivariate analysis of variance. Austral Ecol 26, 32-46, doi:10.1111/j.1442-9993.2001.01070.pp.x (2001).9Chao, A. Estimating the population size for capture-recapture data with unequal catchability. Biometrics 43, 783-791 (1987).10Hill, M. O. Diversity and evenness: a unifying notation and its consequences. Ecology 54, 427-432, doi:10.2307/1934352 (1973).GUARANTOR OF THE ARTICLEGirish Hiremath, MD MPHAUTHOR CONTRIBUTIONS: Study conception and design: GH, SA, HC, SRDCollecting data, analyzing biopsies and saliva samples: GH, MHS, HC, SVR, SRD Generation, analysis, and interpretation of salivary microbiome data: GH, MHS, HB, AT, SVR, SRDCritical revisions of the manuscript: GH, MS, HB, AT, HC, SA, SVR, SRD.FINANCIAL SUPPORTThis study was funded by the American College of Gastroenterology Clinical Research Award. G.H. is supported by American College of Gastroenterology Junior Faculty Career Development Award, Vanderbilt University Turner Hazinski award, Vanderbilt University Katherine Dodd Faculty Scholar program, and the Consortium of Eosinophilic Gastrointestinal Disease Researchers (U54 AI117804) training award. Consortium of Eosinophilic Gastrointestinal Disease Researchers (CEGIR) is part of the Rare Disease Clinical Research Network, an initiative of the Of?ce of Rare Diseases Research, National Center for Advancing Translational Sciences, and is funded through collaboration between the National Institute of Allergy and Infectious Diseases, the National Institute of Diabetes and Digestive and Kidney Diseases, and the National Center for Advancing Translational Sciences. The CEGIR is also supported by patient advocacy groups including the American Partnership for Eosinophilic Disorders, Campaign Urging Research for Eosinophilic Diseases, and Eosinophilic Family Coalition. Vanderbilt Institute for Clinical and Translational Research grant support?(NIH/NCATS UL1 TR000445, U54 RR24975). SD is also supported by NIH-funded Tennessee Center for AIDS Research (P30 AI110527) and U19AI095227. Content is solely the responsibility of the authors and does not represent official views of the CDC and the NIH.POTENTIAL COMPETING INTEREST Andrey Tovchigrechko is employed by MedImmune, the biologics arm of AstraZeneca, and owns AstraZeneca stock. ................
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