cfDNA TSS Enrichment for Accurate Gene Expression Inference
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Solution Overview
Problem
Current whole genome sequencing methods have limited capability to infer expression of individual genes or gene sets due to low depth of coverage, hindering accurate gene expression inference and tissue-of-origin classification, particularly in noninvasive cancer detection.
Innovation Solution
A method involving the preparation of a methylation sequencing library by extracting cell-free DNA, converting unmethylated cytosines to uracils, enriching with hybridization probes targeting transcription start site sequences, amplifying, and determining nucleic acid sequences to calculate gene expression scores, utilizing next-generation sequencing and machine learning for accurate gene expression detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If whole genome sequencing is performed on cfDNA, then comprehensive genomic information is obtained, but the depth of coverage at sites of interest is insufficient for accurate gene expression inference
Solution Approach 1:
The patent extracts and enriches only the relevant cfDNA fragments containing transcription start site sequences using hybridization probes, rather than analyzing the entire genome. This extraction of specific target sequences from the complex cfDNA mixture enables sufficient coverage depth for accurate gene expression inference while reducing the burden of whole genome sequencing.
Solution Approach 2:
The patent changes the sequencing approach from uniform whole genome sequencing to targeted sequencing of enriched TSS regions. By modifying the sequencing parameters to focus on specific genomic locations with higher sequencing depth, the method achieves the coverage necessary for accurate gene expression inference without the limitations of standard WGS.
2Quantity of substance
If hybridization probes are used to enrich cfDNA fragments, then sequencing depth at target sites increases, but the process complexity and time increase
Solution Approach 1:
The patent performs preliminary enrichment of target cfDNA fragments using hybridization probes before sequencing. By conducting this enrichment step in advance, the method ensures that sufficient target material is available for sequencing, achieving the necessary depth of coverage while organizing the complex process into manageable sequential steps.
Solution Approach 2:
The patent introduces hybridization probes as intermediary molecules that mediate between the cfDNA fragments and the sequencing process. These probes selectively bind to target sequences, enabling enrichment and subsequent high-depth sequencing while simplifying the overall workflow compared to direct whole genome sequencing at equivalent depth.
3Reliability
If conventional WGS methods are used, then the process is straightforward, but the ability to detect low-abundance gene expression signals is insufficient
Solution Approach 1:
The patent applies local quality enhancement by concentrating sequencing resources on specific TSS regions rather than distributing them uniformly across the genome. This localized approach to sequencing enables the detection of low-abundance gene expression signals by achieving high depth of coverage at target sites, thereby improving detection sensitivity without sacrificing overall productivity.
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
Enables accurate detection of gene expression with high accuracy, sensitivity, and specificity, facilitating noninvasive cancer detection and minimal residual disease monitoring.
Implementation Method 1
enriching the plurality of converted cfDNA fragments to produce enriched converted cfDNA fragment molecules, wherein the enriching comprises contacting the plurality of converted cfDNA fragments with a probe set comprising hybridization probes having sequence complementarity to at least two transcription start site (TSS) sequences
Implementation Method 2
amplifying the enriched converted cfDNA fragment molecules to produce amplified enriched converted cfDNA fragments
Data Source
AI summary
Methods and systems disclosed herein can improve inference of gene expression using cell-free DNA fragments. In an aspect, the present disclosure provides a computer-implemented method for inferring gene expression, the method comprising: obtaining a biological sample from a subject; extracting cell-free deoxyribonucleic acid (cfDNA) from the biological sample, wherein the cfDNA comprises a plurality of cfDNA fragments; performing a sequencing assay on the plurality of cfDNA fragments to generate a plurality of cfDNA sequencing fragments; computer processing the plurality of cfDNA sequencing fragments; and calculating, based at least in part on the computer processing, a gene expression score for a gene in a plurality of genes, wherein the gene expression score indicates a probability of expression or non-expression of the gene in the plurality of genes.