Single-Stranded cfDNA Library Prep for Transplant Infection Detection

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Solution Overview

Problem

Current methods for diagnosing urinary tract and peritoneal infections, particularly in transplant recipients, are inadequate as they fail to detect shorter, degraded, and single-stranded fragments of cell-free DNA, and are insensitive to viral infections, limiting their ability to provide comprehensive insights into bacterial growth dynamics and antibiotic resistance.

Innovation Solution

A method involving the preparation of single-stranded sequencing libraries from urine or peritoneal dialysis fluid samples, followed by next-generation sequencing, which allows for the detection and analysis of non-human DNA sequences, including microbial DNA, and determination of their source, replication status, and antibiotic resistance, as well as the identification of donor DNA in transplant recipients through alignment with reference genomes and databases.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If standard library preparation methods based on ligation of double strand DNA sequencing adapters are used, then the method is relevant to a wide range of applications and relatively simple to implement, but shorter fragments, highly degraded fragments and partially single-stranded fragments of DNA in circulation remain undetected

Engineering Contradiction:

Improveapplicability to wide range of applicationsVSAvoiddetection sensitivity for diverse DNA fragments

Core Design Contradiction:

Adaptability or versatilityVSMeasurement precision

Solution Approach 1:

The patent changes the fundamental parameter of DNA strand configuration from double-stranded to single-stranded library preparation. This allows the method to detect a broader spectrum of cfDNA fragments including short, degraded, and partially single-stranded fragments that are missed by conventional double-stranded methods, while maintaining broad applicability across different applications

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent inverts the conventional approach by preparing single-stranded libraries instead of double-stranded libraries. This inversion enables detection of DNA fragments that are incompatible with double-stranded ligation methods, including highly degraded and partially single-stranded fragments, thereby improving measurement precision without sacrificing versatility

Inventive Principle:

Principle #13The other way round (Inversion)

2Reliability

If multiple bead-based size-selective steps are used to eliminate unwanted adapter-dimer products, then adapter-dimer contamination is reduced, but the detection of shorter and degraded DNA fragments is compromised

Engineering Contradiction:

Improvepurity of sequencing libraryVSAvoiddetection capability for short and degraded fragments

Core Design Contradiction:

ReliabilityVSMeasurement precision

Solution Approach 1:

The patent changes the size selection parameter from bead-based physical separation to a method that is inherently compatible with short fragments. The single-stranded library preparation method allows recovery of fragments as short as 20-30 base pairs while maintaining library purity through alternative purification strategies that do not rely on size-based bead separation

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent extracts the size-selective step from the library preparation process by using methods that do not require bead-based separation. This extraction allows shorter and degraded fragments to be retained and sequenced while unwanted adapter-dimers are eliminated through other means, resolving the contradiction between purity and detection capability

Inventive Principle:

Principle #2Taking out (Extraction)

3Measurement precision

If in vitro culture methods are used for diagnosis of UTI, then cultivable organisms can be detected, but viral infections and commensal microbiota cannot be detected and bacterial growth dynamics cannot be informed

Engineering Contradiction:

Improvedetection of cultivable organismsVSAvoiddetection range including viruses and microbiota

Core Design Contradiction:

Measurement precisionVSAdaptability or versatility

Solution Approach 1:

The patent applies universality by using next-generation sequencing of cfDNA to simultaneously detect multiple types of pathogens including bacteria, viruses, and fungi, as well as to assess bacterial growth dynamics and antibiotic resistance. This single method replaces multiple specialized tests, achieving both specific detection of cultivable organisms and broad detection of non-cultivable pathogens

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

The patent substitutes the mechanical culture-based detection system with a molecular sequencing-based system. This replacement enables detection of viral infections and commensal microbiota that cannot be cultured, while also providing information on bacterial growth dynamics and antibiotic resistance through sequence analysis

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Data Source

PatentUS20240026456A1Methods of detecting cell-free DNA in biological samples

Publication Date: 2024.01.25 CORNELL UNIVERSITY

AI summary

The present disclosure is directed to methods of detecting cell-free DNA (cfDNA) in biological samples and using it to quantify organ damage and identify pathogens. In some aspects, the biological samples are from patients who have undergone solid-organ transplantation. The disclosure is also directed to methods of detecting and analyzing methylation patterns in cell-free DNA from organ transplant patients to identify the presence of pathogens as well as quantify contributing tissue proportions as a measurement of the host response.