Abstract
AbstractThe overall goal of our Demonstration Project is to address the hypothesis that the gut microbiota plays a critical role in the pathogenesis of inflammatory bowel disease (IBD). To circumvent the confounding effects of mucosal inflammation and medications that independently bias microbial selection, we have set out to study a unique clinical model of ulcerative colitis (pouchitis) where the development and status of the gut microbiota can be assessed prospectively and relative to the outcome of disease. Pouchitis is an inflammatory condition of a surgically-created pseudorectum that is unique to ulcerative colitis and rarely occurs when this procedure is performed for treatment of other non-IBD diseases. Since the incidence of developing pouchitis within one year is over 50%, the likelihood of detecting antecedent changes in microbiota is high. Our multi-center study involves an integrated team of researchers with expertise in clinical manifestations of pouchitis (Univ. Chicago), cultivation of diverse microorganisms (MSU), molecular microbial ecology (MSU/UMich/MBL), and metagenomics (ANL). Our goal is to enroll up to 50 patients with pouchitis who will be followed clinically and endoscopically (sampling from the unprepped pouch) for a period of 18 months each. Though the use of high-throughput, cost-effective technologies, which members of this team have played a key role in developing, we intend to demonstrate a critical role for the gut microbiome in the development of the dysregulated mucosal immune response that is the hallmark of IBD. In order to identify associations between the structure and dynamics of the gut microbial community and specific clinical endpoints, we will scan for markers in the fecal microbial community that are associated with specific, quantifiable measures of disease activity or progression. Collectively, these studies will provide insights into how enteric microbiota cause or contribute to the etiopathogenesis of human ulcerative colitis. This knowledge will lead the way to novel ways to monitor patients with IBD and to guide the development of novel preventative and therapeutic interventions.In accordance with the guidelines of the Human Microbiome Project (HMP), data generated by this study (NCBI Project ID 463154) will be subject to a 12 month publication moratorium from the date of data submission to dbGaP and SRA.
Highlights
The overall goal of our Demonstration Project is to address the hypothesis that the gut microbiota plays a critical role in the pathogenesis of inflammatory bowel disease (IBD)
Pouchitis is an inflammatory condition of a surgicallycreated pseudorectum that is unique to ulcerative colitis and rarely occurs when this procedure is performed for treatment of other non-IBD diseases
Though the use of highthroughput, cost-effective technologies, which members of this team have played a key role in developing, we intend to demonstrate a critical role for the gut microbiome in the development of the dysregulated mucosal immune response that is the hallmark of IBD
Summary
The Marine Biological Laboratory used 454 Life sciences recursive phase algorithm “beta pipeline” to recover an average of 890,000 reads that extend from our proximal primers through “landmark” evolutionary conserved sequences GGATTAGATACCC (E. coli 785-797) for V3 V5 amplicons (455 ± 50nt) or TGGGCGTAAAG (E. coli 565-575) for V6 – V4 amplicons (490nt ± 50nt). Our analyses quarantine and exclude reads that do not exactly match the MIDs (Multiplex Identifiers) or proximal rRNA primers, reads containing one or more ambiguous base calls (Ns), reads that exhibit greater than 2 mismatches to the evolutionary conserved sequences, and reads lacking the “landmark” evolutionary conserved sequences within 50 nt of their predicted position. These quality control procedures result in an average error rate of fewer than one error/ 500 positions. For the shotgun metagenomic data analysis, Argonne National Laboratory’s quality control is implemented through procedures used to include/exclude a particular sequenced read for subsequent analysis:. - Reads may not exhibit ambiguous base calls; those that do are removed from consideration. - Reads may not exhibit ambiguous base calls; those that do are removed from consideration. - Reads that exhibit an uncharacteristic length – with respect to the particular sequencing platform used and/or to the distribution of lengths among all sequenced reads. - Reads are screened to remove those that can be attributed to eukaryotic contamination; they are screened to remove artificial duplicates
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