Host DNA Depletion in Metagenomic Sequencing via RNA Probe Hybridization
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Solution Overview
Problem
Low levels of microbial DNA in human tissues make it costly and inefficient for metagenomic analysis, as existing sequencing techniques require high coverage to detect infectious agents, leading to unsuitable samples due to high host DNA content.
Innovation Solution
A method for isolating microbial DNA from samples containing host DNA using tagged DNA samples, hybridization with affinity-tagged RNA probes, and capture agents to separate microbial DNA from host DNA, allowing for whole-genome sequencing and improved metagenomic analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If shotgun sequencing is performed on samples with high host DNA content, then sequencing coverage increases, but the proportion of microbial DNA sequenced decreases
Solution Approach 1:
The patent extracts and removes host DNA from the sequencing library using host-specific probes that hybridize to host DNA sequences. This allows selective depletion of host DNA while preserving microbial DNA, thereby increasing the proportion of microbial DNA in the final sequenced library without requiring increased sequencing coverage
Solution Approach 2:
The patent uses affinity-tagged RNA probes as intermediaries to selectively bind and capture host DNA. These probes serve as mediators between the host DNA and the capture agent (magnetic beads), enabling specific isolation and removal of host DNA while leaving microbial DNA untouched
2Reliability
If sequencing depth is increased to detect microbial DNA in high host DNA samples, then detection sensitivity improves, but sequencing cost increases
Solution Approach 1:
By extracting and removing host DNA before sequencing, the patent increases the relative concentration of microbial DNA in the library. This allows achieving the same detection sensitivity with lower sequencing depth, thereby reducing sequencing costs while maintaining reliability
3Loss of information
If whole-genome shotgun sequencing is used on samples with low microbial DNA content, then comprehensive metagenomic data is obtained, but sequencing efficiency decreases
Solution Approach 1:
The patent extracts host DNA from the sample before sequencing, thereby concentrating the microbial DNA content in the remaining library. This increases sequencing efficiency by ensuring that a higher proportion of sequenced reads correspond to microbial genomes, while still maintaining comprehensive metagenomic data coverage
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
This method significantly increases the yield of microbial DNA, reducing the proportion of host DNA in sequencing libraries, thereby enhancing the efficiency and cost-effectiveness of metagenomic analysis by isolating and sequencing microbial DNA effectively.
Implementation Method 1
b) hybridizing the extracted DNA, in solution, with affinity-tagged RNA probes generated by in vitro transcribing
Implementation Method 2
c) binding the product of step b) with a capture agent that is tethered to a substrate
Data Source
AI summary
Described herein is a method for isolating microbial DNA from a sample that comprises host DNA and microbial DNA. In some embodiments, the method may comprise: obtaining a tagged DNA sample, wherein the tagged DNA sample contains host DNA and microbial DNA, both comprising an appended universal adaptor; b) hybridizing the extracted DNA, in solution, with affinity-tagged RNA probes generated by in vitro transcribing, in the presence of an affinity-tagged ribonucleotide, a library of fragmented host DNA that has been ligated to an RNA promoter adaptor; c) binding the product of step b) with a capture agent that is tethered to a substrate, in the presence of RNA oligonucleotides that are complementary to or have the same sequence as one or more strands of the universal adaptor, thereby capturing the host DNA on the substrate; and d) collecting the unbound DNA, wherein the unbound DNA comprises the microbial DNA.