Methods and compositions for enriching non-host sequences in host samples
Inventors
Assignees
University of Alaska Fairbanks
Publication Number
US-11035000-B2
Publication Date
2021-06-15
Expiration Date
2034-12-04
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Abstract
Disclosed are compositions and methods for enriching a non-host sequence from a host sample. Also disclosed are compositions and methods for detecting a non-host sequence in a host sample. For example, a pathogen can be enriched and detected in a sample taken from a human without knowing what the pathogen is.
Core Innovation
The invention provides methods and compositions for enriching non-host (e.g., non-human) nucleic acid sequences within a host sample, such as a human clinical specimen, through the use of non-host target primers that are designed to avoid hybridizing to the most abundant host transcripts. These primers selectively amplify non-host nucleic acids, resulting in an enriched population of sequences exogenous to the host, including potential pathogens like viruses, bacteria, and fungi, without prior knowledge of the specific pathogen present.
The disclosed technology addresses the major obstacle in applying next generation sequencing (NGS) for pathogen detection in clinical diagnostics, namely the overwhelming excess of host-derived nucleic acids compared to scarce pathogen sequences. Traditional diagnostic approaches require pre-existing knowledge of the target pathogen and can be slow, costly, and inconclusive when the infection source is unknown. By contrast, the disclosed method enriches for non-host sequences, greatly enhancing the ability to detect pathogens, even when they are unknown or present at extremely low abundance.
The core process involves isolating nucleic acids (DNA, total RNA, or mRNA) from a host sample, selectively amplifying them with non-host target primers (which do not bind to the most abundant host transcripts), and, optionally, performing subtractive hybridization against a reference population of host cDNAs to further deplete host background. The resulting enriched non-host nucleic acids are then detected by sequencing, facilitating comprehensive and unbiased pathogen identification.
Claims Coverage
The patent includes three independent claims that define three main inventive features.
Selective enrichment of non-human nucleic acids using specific non-human target primers
A method for enriching non-human nucleic acids from a human sample by selectively amplifying nucleic acids isolated from the human sample using non-human target primers, where these primers are at least the oligonucleotides in FIG. 10 or FIG. 11.
Selective reverse transcription with non-human target primers complementary to specified oligonucleotides
A method for enriching non-human nucleic acids from a human sample by hybridizing a set of non-human target primers to total RNA or mRNA isolated from the human sample and selectively reverse transcribing the RNA to form an enriched population of non-human cDNA strands, wherein the set of non-human target primers are the complement of at least the oligonucleotides in FIG. 10 or FIG. 11.
Detection of non-host nucleic acids by selective amplification and sequencing with defined primers
A method of detecting non-host nucleic acids in a host sample by (a) selectively amplifying nucleic acids isolated from the host sample using non-host target primers that are at least the oligonucleotides in FIG. 10 or FIG. 11 to form an enriched population of non-host nucleic acids; and (b) detecting the non-host nucleic acids by sequencing the enriched population of unknown, non-host nucleic acids.
In summary, the inventive features establish specific methods for enriching and detecting non-host or non-human nucleic acids by employing carefully selected sets of primers that do not hybridize to abundant host sequences, enabling sensitive and broad-spectrum identification of exogenous sequences in host samples.
Stated Advantages
The disclosed methods greatly increase the presence of pathogenic sequences in clinical samples, allowing for more efficient detection.
The technology enables comprehensive, unbiased diagnosis of pathogens responsible for any infectious disease without prior knowledge of the infectious agent.
By eliminating or reducing abundant host transcripts, sequencing complexity is greatly reduced and enrichment for low-abundance pathogenic sequences is achieved.
The method is more time- and cost-efficient than traditional diagnostic approaches that require numerous specific tests and sample collections.
The approach enables detection of both known and unknown pathogens, improving laboratory response during outbreaks and for cases with unclear diagnoses.
PATHseq can be used as a universal, one-step, and unbiased testing method for improving blood supply safety by detecting known and previously unknown pathogens.
Documented Applications
Diagnosis of infectious diseases in clinical specimens, particularly where the cause is unknown or current diagnostic methods are inconclusive.
Investigation and epidemiological characterization of infectious disease outbreaks using genomic techniques, including rapid identification of pathogens.
Screening blood supplies for pathogens, including both known and unknown agents, to ensure blood safety.
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