Nucleic Acid Labeling with Segmented Barcode Incorporation

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Solution Overview

Problem

Current methods for target enrichment in next-generation sequencing (NGS) face challenges in efficiently labeling nucleic acid libraries with library-specific and molecule-specific barcodes, which are crucial for multiplex sequencing processes to accurately identify and quantify unique DNA molecules and mutations.

Innovation Solution

The method involves contacting polynucleotide libraries with primers that hybridize to adapter sequences, extending them with polymerases to incorporate sample-specific and molecule-specific barcodes, and then amplifying these products using PCR or other methods, allowing for the incorporation of variable barcode sequences for unique identification.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If target enrichment methods are used to increase relative abundance of target sequences, then sequencing cost and data analysis burden are reduced, but compatibility with multiplex sequencing processes becomes challenging

Engineering Contradiction:

Improvesequencing cost efficiencyVSAvoidcompatibility with multiplex sequencing

Core Design Contradiction:

ProductivityVSAdaptability or versatility

Solution Approach 1:

The labeling process is segmented into two distinct stages: first adding molecule-specific barcodes (UIDs) to individual DNA molecules before amplification, then adding library-specific barcodes (MIDs) to the amplified library. This segmentation allows each enrichment method to be optimized independently while maintaining compatibility with multiplex sequencing workflows.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Molecule-specific barcodes are incorporated into DNA molecules during the target enrichment process before amplification occurs. This preliminary action ensures that unique molecular identifiers are present on original template molecules, enabling accurate duplication tracking while maintaining compatibility with subsequent multiplex sequencing steps.

Inventive Principle:

Principle #10Preliminary action

2Measurement precision

If molecule-specific barcodes are added prior to amplification, then unique DNA molecules can be distinguished from PCR duplicates, but the labeling process becomes more complex

Engineering Contradiction:

Improveidentification accuracy of unique moleculesVSAvoidlabeling process complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

A specialized primer is used as an intermediary to transfer the molecule-specific barcode from the template DNA to the amplified products. This primer contains the barcode sequence and facilitates its incorporation during the amplification process, simplifying the overall labeling procedure while maintaining measurement precision.

Inventive Principle:

Principle #24Intermediary (Mediator)

3Adaptability or versatility

If library-specific barcodes are added through PCR primers, then multiple libraries can be combined for sequencing, but the barcode incorporation efficiency may vary

Engineering Contradiction:

Improvemultiplex sequencing capabilityVSAvoidbarcode incorporation consistency

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The PCR amplification conditions are optimized with specific parameters including 98°C denaturation for 10 seconds, 60°C annealing for 30 seconds, and 72°C extension for 30 seconds, repeated for 15 cycles. These controlled parameter changes ensure consistent and efficient barcode incorporation across all libraries while maintaining multiplex sequencing capability.

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 enables accurate identification and quantification of unique molecules in nucleic acid samples, facilitating multiplex NGS processes by enhancing the labeling efficiency of nucleic acid libraries with molecular barcodes, thereby improving diagnostic capabilities.

Implementation Method 1

contacting the library of polynucleotide molecules with primers (e.g., sample index primers) comprising a first hybridization sequence (i.e., a sequence capable of hybridizing to at least a portion of the 3′ adapter)

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

incubating the hybridized polynucleotide molecules with a polymerase such that the polymerase extends 3′ end of the primers (e.g., sample index primers) to form primer extension products

Methodology Applied

Scientific EffectDNA synthesis:

Data Source

PatentUS10246702B1Compositions and methods for labeling target nucleic acid molecules

Publication Date: 2019.04.02 NEW ENGLAND BIOLABS INC

AI summary

Provided herein are methods and compositions for labeling target nucleic acid molecules with molecular barcodes.