cfDNA Gene Expression Analysis via TSS Read Depth
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Solution Overview
Problem
Current methods for measuring gene expression from cell-free DNA (cfDNA) are limited by requiring high tumour purity and are not sensitive enough to detect dynamic changes in gene expression across all genomic regions, making them inaccurate for monitoring cancer treatment effects.
Innovation Solution
A method involving sequencing cfDNA fragments, aligning them with a reference genome, identifying reads aligned to gene transcriptional start sites, determining read depth, and generating corrected read depth to categorize gene expression levels, allowing for dynamic assessment of gene expression over time.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If previously developed methods using ctDNA fragmentation patterns are used to determine gene expression, then binary determination of gene expression can be achieved, but high tumour purity is required and the method is not sensitive enough to detect dynamic changes in gene expression
Solution Approach 1:
The invention changes the measurement parameter from binary expression determination to continuous read depth analysis. By quantifying the actual number of reads mapping to TSS regions and comparing them to control regions, the method transitions from categorical (expressed/unexpressed) to quantitative measurement, enabling detection of dynamic changes and improving sensitivity under low tumour purity conditions
Solution Approach 2:
The invention introduces control genomic regions as an intermediary reference system. By comparing TSS region read depths against control region read depths within the same sample, the method compensates for variations in tumour purity and cfDNA input, thereby improving measurement reliability without requiring high tumour purity
2Ease of operation
If ctDNA is used to infer gene expression profiles, then non-invasive cancer detection and monitoring is enabled, but the method is limited to genes with high-amplitude copy number amplifications
Solution Approach 1:
The invention uses control genomic regions as intermediary references to normalize TSS read depths. This internal control system allows the method to function across all genomic regions regardless of copy number status, removing the limitation to only high-amplitude amplification regions while preserving the non-invasive nature of plasma sampling
Solution Approach 2:
The invention changes from relying on copy number amplification signals to using relative read depth ratios between TSS and control regions. This parameter transformation enables the method to detect gene expression changes across all genomic regions, not just those with high-amplitude amplifications, thereby improving versatility
3Measurement precision
If standard RNA sequencing is used as the gold standard for gene expression measurement, then accurate gene expression profiles are obtained, but invasive tissue biopsy is required which limits serial monitoring
Solution Approach 1:
The invention creates a molecular copy relationship where cfDNA fragmentation patterns serve as a proxy for RNA expression profiles. By establishing that TSS-proximal cfDNA read depths correlate with gene expression levels, the method uses DNA fragments as copies that reflect transcriptional activity, enabling non-invasive measurement with accuracy comparable to RNA sequencing
Solution Approach 2:
The invention introduces cfDNA as an intermediary molecule that bridges the gap between invasive RNA measurement and non-invasive DNA measurement. The cfDNA fragmentation patterns act as mediators that carry expression information from the tumour to the plasma, allowing indirect but accurate measurement without tissue biopsy
Data Source
AI summary
The present disclosure relates generally to methods for the analysis of gene expression. In particular, the methods of the present disclosure are based on the measurement of gene expression from fragments of cell-free DNA (cfDNA), which is useful in non-invasive methods for monitoring disease status in cancer patients and in methods for the treatment of cancer patients.