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Methods for Characterizing Unculturable Microbiome Components

IP.com Disclosure Number: IPCOM000199347D
Publication Date: 2010-Aug-30
Document File: 7 page(s) / 145K

Publishing Venue

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Abstract

The bacterial species composition of microbial communities, or microbiomes, is of growing interest to biologists. The National Institutes of Health has recently started a Human Microbiome Project to investigate the numerous microbes that live in various environments on and in the human body, and to look at changes in the microbiome in health and disease. However, studies of the human microbiome, or any other microbiome, are difficult because not all the organisms in the microbiome are known, and many of those organisms cannot be cultured. The methods described below provide way to investigate previously un-isolated microbial components of a microbiome.

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Methods for Characterizing Unculturable Microbiome Components

ACH, ROBERT

Scheffer-Wong, Alicia F.

Method 1: Use of OLS Libraries and Oligo-FISH to Isolate

Unculturable Microbiome Components

The bacterial species composition of microbial communities, or microbiomes, is of growing interest to biologists.  The National Institutes of Health has recently started a Human Microbiome Project to investigate the numerous microbes that live in various environments on and in the human body, and to look at changes in the microbiome in health and disease.  However, studies of the human microbiome, or any other microbiome, are difficult because not all the organisms in the microbiome are known, and many of those organisms cannot be cultured.  The methods described below provide way to investigate previously un-isolated microbial components of a microbiome.

The general steps in the method are set forth below:

            1. Isolate a biological sample

            The first step is to isolate a biological sample containing organisms from a microbiome. For example, one can obtain a skin scraping from the heel, toe web, umbilicus, inginual crease, interdigital web space, manurbium, nase, glabell, auxillary vaule, or a biopsy, lavage or wash of an intestinal lining or nasal passage, for example.

            2. Sequence

DNA

from the sample

            The second step is to isolate

DNA

from the sample and perform metagenomic sequencing using a high throughput sequencing method.  Metagenomic sequencing is the sequencing of total

DNA

from an environmental sample.  The organisms whose

DNA

is being sequenced is either known or unknown. This step can be performed using one of many available sequencing technologies, including those of 454 Life Sciences (Roche), Illumina, Helicos, and Pacific Biosciences.

            3. Remove sequences of known origin from the sequence data

            In the third step of the method, the sequences of known origin are removed from the sequence data. For example, if the sample is from a human microbiome, all human

DNA

sequences are computationally removed from the sequence data.  Next, all sequences from known (i.e. previously sequenced) microbes (or any other type of organism) are computationally removed.  The remaining

DNA

sequence will be of unknown origin, i.e. from previously uncharacterized microbes or other organisms. This step can be done using known computational techniques, including BLAST, FASTA and other sequence alignment programs.

            4. Produce oligonucleotides directed to sequences of unknown origin

In the fourth step of the method, oligonucleotides that hybridize with the sequences of unknown origin are made, and then fluorescently labeled. In certain cases, the oligonucleotides may be synthesized in an array using in situ synthesis methods in which nucleotide monomers are sequentially added to a growing nucleotide chain that is...