Targeted sequencing of genetic regions that differ between two DNA preparations; used to study genetic variation among closely related species and microbial communities; for identifying sources of fecal pollution
Inventors
Shanks, Orin C. • Domingo, Jorge Santo • Graham, James E. • Lu, Jingrang
Assignees
United States, As Represented By Us Environmental Protection Agency • US Environmental Protection Agency
Publication Number
US-8058000-B2
Publication Date
2011-11-15
Expiration Date
2025-12-27
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
Targeted sequencing of genetic regions that differ between two DNA preparations uses genomic fragment enrichment. This method can be used to study genetic variation among closely related species and microbial communities, particularly for identifying sources of fecal pollution.
Core Innovation
The invention provides a method called Genome Fragment Enrichment (GFE) that uses solution phase competitive DNA hybridization to identify microbial DNA sequences that are unique or divergent between two DNA preparations. This targeted sequencing approach enriches genomic fragments present in one DNA community but absent or significantly different in another. The method is especially applicable to studying genetic variation among closely related microbial species and complex microbial communities, such as fecal samples from different animal species.
The primary problem addressed by the invention is the difficulty in specifically detecting sources of fecal pollution in environmental waters. Existing microbial water quality methods rely on culturable fecal indicator bacteria that cannot discriminate among different animal sources or bacterial strains. Prior genotypic methods, such as ribotyping and rep-PCR, are limited by the need for large reference databases and by secondary habitat populations that confound source identification. Moreover, most existing PCR-based microbial source tracking (MST) methods target 16S rDNA genes, which exhibit limitations in specificity and sensitivity, and there has been a lack of library-independent, non-16S rDNA assays for certain sources like chicken fecal pollution. Thus, there is a need for a method that can identify DNA sequences encoding proteins directly involved in host-microbe interactions, with high levels of host-specific genetic variation, to improve MST.
The invention solves this problem by using the GFE method to enrich DNA segments that differ between complex microbial communities, such as fecal metagenomes from various animals (e.g., chicken, pig, cow, human). The method employs competitive hybridization of labeled DNA fragments from a tester community with DNA fragments from a blocker community to isolate unique DNA sequences. The enriched sequences are then cloned, sequenced, and used to develop specific PCR primer sets capable of differentiating microbial species, strains, and animal sources of fecal pollution. The approach applies both to pure bacterial genomes (e.g., differentiating Enterococcus faecalis from E. faecium) and complex metagenomes, and can identify genetic markers for environmental monitoring, risk assessment, and microbial ecology studies.
Claims Coverage
The patent contains two independent claims focused on methods for identifying differences between microbial communities and microbial genomes using competitive DNA hybridization and sequence enrichment.
Method for identifying differences between microbial communities
Obtaining genomic DNA from fecal samples of two groups of animals, creating composites designated tester and blocker, labeling the tester DNA, performing competitive hybridization between tester and blocker DNA fragments, incubating with additional tagged tester DNA fragments to form DNA hybrids, capturing these hybrids containing terminal tags, amplifying only tagged fragments by PCR, enriching sequences unique to the tester community, and identifying the enriched sequences to distinguish microbial community differences.
Method for identifying genetic differences between two microbial genomes
Obtaining labeled genomic DNA fragments from a first microorganism and hybridizing them with fragments from a second microorganism; incubating these with additional fragments of the first microorganism containing defined terminal sequence tags to form DNA hybrids; capturing the hybrids with tags; PCR amplification of only tagged fragments; obtaining enriched sequences unique to the first microorganism; and identifying these sequences to recognize genomic differences.
The claims cover methods employing competitive solution hybridization and positive selection of labeled DNA fragments to enrich and identify unique genetic sequences distinguishing microbial communities or microbial genomes, enabling development of specific detection assays.
Stated Advantages
The invention provides a positive selection method that rapidly and efficiently identifies numerous species-, strain-, and host-specific microbial DNA sequences for developing specific PCR assays to discriminate fecal pollution sources.
Unlike prior negative subtraction or PCR-based methods, GFE reduces false positives and dependence on large reference databases, enriching for genomic regions encoding functional proteins involved in host-microbe interactions with greater genetic variation and specificity.
The method is applicable to complex metagenomes without culturing microorganisms, allowing identification of novel genetic markers even from uncultured bacteria, enhancing microbial source tracking and environmental monitoring.
GFE enables design of highly specific and sensitive PCR primers and probes for presence/absence detection and real-time quantification, facilitating accurate assessment of fecal pollution sources relevant to water quality regulation and risk management.
Documented Applications
Studying genetic variation among closely related bacterial species or strains via comparative microbial genome analysis.
Identifying species-specific, strain-specific, and host-specific microbial DNA sequences from animal fecal metagenomes for microbial source tracking (MST) to determine origins of fecal pollution in environmental watersheds.
Developing PCR primer sets for use in presence or absence detection, real-time PCR assays, and microarray applications to detect and quantify animal-specific fecal contamination.
Environmental monitoring and risk assessment of water quality in natural surface and ground waters susceptible to fecal contamination from animals such as cattle, chicken, pigs, humans, and birds.
Ecological studies of microbial communities, discovery of novel virulence factors, and studying host-microbial interactions in gastrointestinal tracts of various animals.
Interested in licensing this patent?