Microbiome and Inflammation Core
Gary B. Huffnagle, Ph.D.
Vincent B. Young, M.D., Ph.D.
Kathryn A. Eaton, D.V.M., Ph.D.
Gnotobiotic Program Manager
Patrick D. Schloss, Ph.D.
Nucleic Acid Isolation
Total DNA and RNA from samples will be isolated using a Roche MagNA Pure LC 2.0 robotic system. This system can process samples from a wide variety of sources (whole blood, leukocytes, bacteria, tissue, etc.). It provides fast isolation, a high degree of reproducibility, and high quality nucleic acid samples that can be used for a variety of PCR applications ranging from T-RFLP analysis to shotgun pyrosequencing.
Terminal Restriction Fragment Length Polymorphism (T-RFLP) Analysis
The microbial community of the mucosal-associated microbiota will first be examined using T-RFLP analysis of 16S rRNA-encoding gene sequences. This provides a quick, cost effective molecular monitoring of changes within the dominant species of the bacterial microbiota. The 16S rRNA gene will be amplified by PCR, using the previously reported primers 8F and 1492R with the 8F primer labeled with a fluorescent tag (5-carboxyfluorescein (5-FAM)). This will result in fluorescent labeling of the PCR product. After amplification of the template DNA, the PCR products will be purified using the Illustra™ GFX™ PCR DNA purification kit. The PCR products will be digested with a restriction endonuclease (in our preliminary studies, we used MspI) and re-purified. The products will then be separated on an automated capillary sequencer. The fluorescent label will be found on the PCR product that is closest to the labeled primer after digestion with the restriction endonuclease (i. e. the terminal restriction fragment, TRF). The automated sequencer will separate, detect, and size each labeled TRF from a 16S rRNA gene amplicon. This analysis is useful when global changes in a microbial community need to be profiled without as much attention to discovering novel phylotypes. For example, we used T-RFLP analysis to profile how infection with the pathogen H. hepaticus altered the indigenous microbiota of mice. A key advantage of the T-RFLP method is that global community profiling can be accomplished at relatively low cost.
16S Clone Libraries
While T-RFLP analysis can provide a broad overview of community structure, knowledge of specific phylotypes is often desired in microbial community analysis. For this purpose, the construction of 16S clone libraries is often employed. To generate 16S clone libraries, unlabeled broad-range 16S primers will be used to amplify DNA samples. Following purification, the PCR products will be ligated into a T-tailed plasmid vector (pCR 2.1; Invitrogen) and will be transformed into an E. coli cloning strain. For each sample, at least 96 clones will be picked for analysis. Plasmids will be purified and sequenced by the DNA Sequencing Core at the University of Michigan. The clone sequences will be curated to remove chimeras and analyzed using a “16S rDNA sequence pipeline” that is integrated into Schloss’s mothur software package. Sequence analysis will begin with the retrieval of raw sequence data and then these data will be moved through a series of software-based quality control tests including base-calling via PHRED and sequence quality assessment and vector trimming with LUCY. Analyses will culminate in phylogenetic and statistical analyses of the clone libraries. One advantage of this approach is that it allows multiple threads of analytical output, and given the modular nature of the pipeline, different analyses can be incorporated as the need arises or as new analytical tools are developed.
The development of “next-generation” sequencing platforms has revolutionized many genomic-based analyses. In the field of microbial ecology, these technologies have been leveraged to increase the depth of coverage of 16S rRNA-encoding gene analysis, to increase speed of genomic sequences and to obtain metagenomic sequences (“metagenomics” in this setting referring to the sum total of the coding capacity of a given microbial community). The pyrosequencing described in this proposal will be carried out on the Roche 454 GS-FLX pyrosequencer at the University of Michigan DNA Sequencing Core. For Variable regions within 16S rRNA genes will be amplified using universal primers fused to the 454 Roche A or B sequencing adapter sequences. Greater than 500,000 individual 16S DNA sequences can be analyzed during one run of the machine and an additional tagging of the PCR primers with 6-8 nucleotide "bar codes" will allow many samples to be analyzed during the run. The quality of PCR amplicons for 454 sequencing will be assessed on a Bioanalyzer 2100 using a DNA1000 LabChip. In addition, shotgun metagenome sequences can be obtained by ligating the sequencing adapters to sheared genomic DNA followed by pyroseqencing. The resulting sequence data can be analyzed using the mothur software using phylotype and OTU-based approaches as well as bioinformatics tools such as MG-RAST, SEED, BLAST to describe and compare the phylogenentic and functional composition of the microbial communities.
Expression of inflammatory/regulatory cytokines and genes
Both custom-assembled QPCR cytokine arrays and pre-made inflammatory and regulatory gene arrays (SABiosciences) will be available. mRNA will be purified from tissues or dispersed cells using Trizol (according to manufacturers protocol), followed by reverse-transcription to cDNA, and addition of the cDNA to the custom PCR array. The real-time PCR will be performed using a Lightcyler 480 (Roche) available in the Microbiome & Inflammation Core. For genes not included on the array, quantitative real-time RT-PCR these using Taqman (hydrolysis) probes and primers obtained from Applied Biosystems (Foster city, CA) or Roche Applied Sciences (Indianapolis, IN). cDNA conversion, amplification, and data analysis will be performed on a Mx3000P real-time PCR system computerized thermocycler from Stratagene (La Jolla, CA), using a Taqman Universal PCR Master mix (Applied Biosystems). Threshold values will be normalized to GAPDH mRNA. Data will be expressed as relative fold increase of specific mRNA (fluorescence intensity) of the treated samples compared with the untreated control sample used as calibrator.
One avenue to an improved understanding of the physiological ecology of the microbiome and its impact on the host is through the study of bacterial isolates in pure culture and in defined mixed cultures. We have experience in 1) routine microbiological plating on selective media for bacteria and fungi, 2) culturing fastidious, anaerobic and extremely oxygen sensitive (EOS) microbes, and 3) using procedures for microbial enrichment and isolation with molecular biological methods to obtain cultures of as yet-unexplored genetic and physiological diversity in the microbiome of the human GI tract. In addition to the culture facilities listed below, training in microbial cultivation techniques will be available through the M&I Core.
Germ-free & Gnotobiotic Mouse Facilities
The germ free mouse resource at the University of Michigan is directed by Dr. Kathryn Eaton. Mice are housed in soft-sided plastic isolators, in which they remain free of all bacteria, exogenous viruses, fungi, and parasites (determined by regular fecal monitoring and periodic control necropsies). Germ-free breeding colonies of mice are maintained in this facility and experimental isolators are used to maintain the germ-free or gnotobiotic status of the mice during experiments. For colonization studies, five week old germ-free mice can be inoculated with a suspension of a specific microbe (mono-colonization), a defined finite group of microbes, or a polymicrobial mixture. Microbes are introduced directly into the stomach with a 24-gauge ball-tipped gavage needle. We have successfully used this procedure in our gnotobiotic facility numerous times to conventionalize germ free mice such that conventionalized microbiota retains its similarity to the donor inoculum microbiota.
Bioinformatics Consultation and Training
Assistance with bioinformatics analysis, consultation and/or training is now part of the Microbiome & Inflammation Core under the direction of our Bioinformatics Specialist, Dr. Patrick Schloss. He developed a suite of bioinformatics tools to describe and compare the richness, membership, and structure of microbial communities using peptide fragment sequences extracted from metagenomic sequence data. Dr. Schloss, in collaboration with a number of other investigators, developed an analysis package that is an open source, platform-independent, community-supported software for describing and comparing microbial communities (mothur). It builds upon previous tools to provide a flexible and powerful software package for analyzing sequencing data and can trim, screen, and align sequences, calculate distances, assign sequences to OTUs, and describe the alpha- and beta-diversity of samples previously characterized by pyrosequencing of 16S rRNA gene fragments (the analysis of more than 222,000 sequences can be completed in less than 2 hours using a laptop computer).