Host Nucleic Acid Depletion for Pathogen Sequencing Enrichment
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Solution Overview
Problem
Current methods for pathogen detection in clinical samples, such as blood and sputum, are hindered by the overwhelming presence of host nucleic acid, which complicates next-generation sequencing (NGS) and leads to suboptimal performance and high costs, as existing depletion techniques are laborious and incomplete.
Innovation Solution
A method involving the use of saponin, DNase, and salts like NaCl/KCl to form a reaction mix at specific concentrations, followed by incubation, effectively depletes host nucleic acid, allowing for rapid and selective enrichment of pathogen nucleic acid for sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional host DNA depletion methods are used, then some host DNA removal is achieved, but the depletion is incomplete and laborious
Solution Approach 1:
The patent combines host cell lysis and host DNA degradation into a single sequential workflow. Saponin is used to lyse host cells and release host DNA, followed immediately by DNase treatment to degrade the released host DNA. This merging of functions into one protocol achieves complete host DNA depletion (≥105-fold) while maintaining pathogen DNA integrity, resolving the contradiction between depletion completeness and operational simplicity.
2Adaptability or versatility
If shotgun metagenomic sequencing is applied to infection diagnosis, then comprehensive pathogen detection is achieved, but the large amount of human DNA present (108:1 to 109:1 ratio) complicates the sequencing
Solution Approach 1:
The patent extracts and removes the problematic host DNA component from the sequencing mixture through selective lysis of host cells using saponin followed by DNase degradation. This extraction of the interfering element (host DNA) reduces the human:pathogen DNA ratio from 108:1 to 109:1 down to at least 105:1, enabling effective shotgun metagenomic sequencing for comprehensive pathogen detection without the overwhelming background of host DNA.
3Quantity of substance
If separate host cell lysis and host nucleic acid degradation steps are deployed, then thorough depletion is achieved, but the process becomes relatively slow
Solution Approach 1:
The patent applies preliminary action by using saponin to lyse host cells and release host DNA before applying DNase for degradation. This preliminary lysis step prepares the host DNA in an accessible form that can be rapidly degraded by DNase, achieving thorough host nucleic acid depletion (≥105-fold) while minimizing total processing time. The sequential but integrated nature of these steps prevents time loss while ensuring complete depletion.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method achieves a high fold depletion of host DNA, up to 105-fold, enabling effective pathogen nucleic acid enrichment, suitable for sequencing-based diagnostics, reducing time and cost while maintaining the integrity of non-host nucleic acid.
Implementation Method 1
adding to said sample a saponin, a DNase, and NaCl and/or KCl
Implementation Method 2
adding to said sample a saponin, a DNase, and NaCl and/or KCl
Implementation Method 3
wherein the addition of said NaCl and/or KCl is sufficient to ensure a final concentration of NaCl and/or KCl in the reaction mix of at least 0.2M
Data Source
AI summary
Provided are methods, compositions and kits for depleting host nucleic acid in a biological sample, said sample having been previously obtained from an animal host.