Barcoded Solid Supports for Haplotype Phasing

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Solution Overview

Problem

Current methods for haplotype phasing, such as those involving hydrogels or barcoded transposons, require long DNA molecules that are cumbersome to isolate, especially from preserved samples like FFPE samples, making it difficult to recover DNA for cancer diagnostic applications.

Innovation Solution

The production of barcoded solid supports through rolling circle amplification (RCA) of circular nucleic acid templates with stem-loop structures, which are disposed on solid supports to facilitate the attachment of target nucleic acids, enabling efficient haplotype phasing and genome analysis.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If long DNA molecules are used for haplotype phasing, then the accuracy of haplotype identification is improved, but the difficulty of isolating and recovering DNA from preserved samples increases

Engineering Contradiction:

Improvehaplotype identification accuracyVSAvoidDNA isolation difficulty

Core Design Contradiction:

Measurement precisionVSEase of manufacture

Solution Approach 1:

The patent segments the long DNA molecule into smaller fragments that can be independently processed and attached to barcoded solid supports. This allows the use of shorter, easier-to-isolate DNA fragments while maintaining haplotype phasing capability through barcode linkage of fragments that originated from the same long molecule.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent introduces barcoded solid supports as intermediary carriers that link DNA fragments to barcodes. These solid supports serve as mediators between the DNA fragments and the sequencing process, enabling haplotype phasing without requiring the isolation of long intact DNA molecules.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If physical separation by droplet formation is used to ensure unique barcode coupling, then the reliability of barcode assignment is improved, but the device complexity and processing time increase

Engineering Contradiction:

Improvebarcode assignment reliabilityVSAvoidprocessing complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent uses multiple copies of barcoded solid supports in a pooled library instead of physical separation of individual molecules. The barcodes are distributed across many solid support copies that can be processed together, eliminating the need for complex droplet-based physical separation while maintaining unique barcode assignment through statistical and computational methods.

Inventive Principle:

Principle #26Copying

3Adaptability or versatility

If FFPE samples are used as DNA source, then the availability of sample types is improved, but the quality and recoverability of DNA decreases

Engineering Contradiction:

Improvesample type availabilityVSAvoidDNA quality

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent changes the parameter of DNA fragment length requirements and uses optimized attachment chemistry that works with degraded FFPE DNA. By adjusting the methodology to accommodate shorter, fragmented DNA while maintaining barcode linkage capabilities, the system can effectively process FFPE samples that would traditionally be considered unsuitable.

Inventive Principle:

Principle #35Parameter changes

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 approach allows for the efficient identification of unique haplotypes and genome assembly from various sample types, including FFPE samples, by providing a method to attach target nucleic acids to barcoded solid supports, enhancing the recovery and analysis of genetic information.

Implementation Method 1

producing a concatemer by rolling circle amplification (RCA) of a circular nucleic acid template

Methodology Applied

Scientific EffectRolling circle amplification:

Implementation Method 2

a stem-loop structure formed from the stem-loop forming region

Methodology Applied

Scientific EffectStem-loop structure formation:

Data Source

PatentUS20230279489A1Barcoded Solid Supports and Methods of Making and Using Same

Publication Date: 2023.09.07 RGT UNIV OF CALIFORNIA

AI summary

Aspects of the present disclosure include methods of making barcoded solid supports. In some embodiments, the methods include producing a concatemer by rolling circle amplification (RCA) of a circular nucleic acid template, where the circular nucleic acid template includes a barcode and a stem-loop forming region, and where the concatemer includes a plurality of linked units, each unit including the barcode and a stem-loop structure formed from the stem-loop forming region. Such methods further include disposing the concatemer on a solid support to produce a barcoded solid support including a plurality of the stem-loop structures extending from the surface of the solid support. The methods may further include treating the stem-loop structures with an agent that produces stem structures having ends compatible with target nucleic acids, and attaching the target nucleic acids to the stem structures. Barcoded solid supports and methods of using the barcoded solid supports are also provided.