Compositions and methods for enriching populations of nucleic acids
Inventors
Blauwkamp, Timothy A. • Christians, Fred • Vilfan, Igor D. • Smith, Scott • Kertesz, Michael
Assignees
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Publication Number
US-11111520-B2
Publication Date
2021-09-07
Expiration Date
Abstract
This disclosure provides methods and compositions that are useful for enriching a particular population of nucleic acids (a “population of interest”) within a complex mixture of nucleic acids. The population of interest may make up a minor portion of a complex mixture of nucleic acids. The methods and compositions provided herein are useful for detecting, predicting, diagnosing, or monitoring a disease or disorder, particularly a disease or disorder caused by a foreign microbe or pathogen.
Core Innovation
The invention relates to generation and use of non-host-targeting oligonucleotide collections for enriching microbial cell-free nucleic acids from host samples. It addresses the problem of obtaining microbial or pathogen nucleic acids from host samples containing host genomic DNA and host cell-free nucleic acids in a way that favors microbial signal over host background.
A core aspect is computational design or synthesis of heterogeneous non-host oligonucleotide pools and their use to reduce host background. Host sequence depletion is performed by hybridization to excess labeled or biotinylated host genomic DNA using blocker oligonucleotides, with labeled host DNA removed by streptavidin beads and/or by size or affinity separation, thereby enriching for microbial nucleic acids in the resulting collection.
Another core aspect is production of high-diversity non-host primer pools using ultramer oligonucleotides. The ultramers are digested with UDG plus endonucleases for abasic sites to create primer pools suitable for downstream capture, priming, PCR, cDNA synthesis, sequencing, and pull-down capture using labeled oligos and/or solid supports.
The disclosure also describes enrichment strategies beyond oligo hybridization, including self-hybridization kinetics, nucleosome depletion using anti-histone or nucleosome antibodies, and length-interval enrichment or depletion for cell-free DNA. These approaches support hypothesis-free pathogen detection, genotyping, monitoring, and microbe identification in a host.
Claims Coverage
Independent claim coverage includes two independent methods. One method enriches microbial cell-free nucleic acids relative to host cell-free nucleic acids using nucleic-acid length ranges where the microbial/host ratio is favored. The other method identifies microbes in a host using enrichment that excludes nucleosomal length ranges, followed by analyzing the enriched nucleic acids and identifying microbes.
Microbial enrichment by favored nucleic acid length ranges
A method of enriching microbial cell-free nucleic acids relative to host cell-free nucleic acids by identifying nucleic acid length ranges at which a ratio of microbial cell-free nucleic acid to host cell-free nucleic acid is favored, providing a host sample comprising both host cell-free nucleic acids and microbial cell-free nucleic acids, and enriching the microbial cell-free nucleic acids by enriching for nucleic acid length ranges at which the ratio is favored.
Microbe identification using non-nucleosomal length-range enrichment
A method of identifying microbes in a host by providing a sample from the host, enriching the sample for microbial cell-free nucleic acids relative to host cell-free nucleic acids such that the enriching comprises enriching for nucleic-acid length ranges that are not nucleosomal length ranges, analyzing the microbial cell-free nucleic acids, and identifying microbes in the host based on the analyzed microbial cell-free nucleic acids.
Across the independent claims, the inventive focus is enriching microbial cell-free nucleic acids using nucleic-acid length ranges, either by selecting ranges where the microbial/host ratio is favored or by enriching for ranges that are not nucleosomal length ranges, followed by analysis to enable microbe identification.
Stated Advantages
Enriches microbial cell-free nucleic acids relative to host cell-free nucleic acids using nucleic-acid length ranges at which the microbial/host ratio is favored.
Enables identification of microbes in the host based on analyzed microbial cell-free nucleic acids enriched using length ranges that are not nucleosomal length ranges.
Enables detecting, diagnosing, and monitoring infectious diseases and multi-drug resistant pathogens based on microbial or pathogen nucleic acids in host cell-free samples.
Documented Applications
Hypothesis-free pathogen detection.
Genotyping.
Monitoring.
Genotyping/antibiotic resistance markers.
Microbe identification in a host based on analyzed microbial cell-free nucleic acids.
Detecting, diagnosing, and monitoring infectious diseases and multi-drug resistant pathogens based on microbial or pathogen nucleic acids in host cell-free samples.
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