Double-tagged oligonucleotide DNA library preparation

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Solution Overview

Problem

Current methods for preparing DNA libraries for Next Generation Sequencing, such as PCR-free, Mate Pair, and single-stranded library preparations face challenges like sequence bias, low yield, and complex workflows due to random ligation and multiple enzymatic steps, which affect the accuracy and efficiency of sequencing results.

Innovation Solution

The use of a double-tagged oligonucleotide (DTO) with pre-selected sequence tags and a linker that provides a breaking site or stopping sequence, allowing for efficient and selective addition of tags to DNA fragments, reducing the need for PCR amplification and simplifying the library preparation process.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If PCR amplification is used to selectively amplify DNA fragments with different tags, then the yield of di-tagged DNA libraries is improved, but sequence bias is introduced affecting accuracy

Engineering Contradiction:

Improveyield of di-tagged DNA librariesVSAvoidsequence accuracy

Core Design Contradiction:

ProductivityVSMeasurement precision

Solution Approach 1:

The invention extracts and eliminates the harmful PCR amplification step from the library preparation workflow. By using a tagmentation-based approach where Tn5 transposase directly inserts sequencing adapters into fragmented DNA, the method achieves sufficient library yield without requiring subsequent PCR amplification, thereby preventing sequence bias while maintaining productivity

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention replaces the enzymatic PCR amplification mechanism with a tagmentation mechanism using Tn5 transposase. This substitution allows for direct insertion of sequencing adapters during DNA fragmentation, eliminating the need for separate PCR amplification steps and avoiding the sequence bias that would otherwise be introduced

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Ease of operation

If multiple enzymatic steps and cleaning steps are performed for mate pair library preparation, then the complexity of the workflow is reduced, but the yield of mate pair DNAs decreases

Engineering Contradiction:

Improveworkflow simplicityVSAvoidyield of mate pair DNAs

Core Design Contradiction:

Ease of operationVSProductivity

Solution Approach 1:

The invention merges multiple separate enzymatic steps (DNA fragmentation, end repair, adapter ligation) into a single tagmentation step using Tn5 transposase. This consolidation simplifies the workflow by reducing the number of cleaning steps and enzyme reactions, while simultaneously improving yield by minimizing sample loss during processing

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The invention performs the adapter insertion action preliminarily during the DNA fragmentation step itself, rather than as a separate subsequent step. By pre-inserting sequencing adapters while fragmenting the DNA, the method eliminates the need for later ligation steps and cleaning operations, thereby simplifying the workflow and preserving sample yield

Inventive Principle:

Principle #10Preliminary action

3Manufacturing precision

If biotinylated nucleotides are used for labeling ends of large DNA fragments, then the specificity of end labeling is improved, but the ligation efficiency decreases

Engineering Contradiction:

Improveend labeling specificityVSAvoidligation efficiency

Core Design Contradiction:

Manufacturing precisionVSProductivity

Solution Approach 1:

The invention replaces the biotinylated nucleotide labeling and ligation mechanism with a tagmentation mechanism using Tn5 transposase. This substitution eliminates the ligation step entirely, as adapters are directly inserted during fragmentation, thereby resolving the contradiction between labeling specificity and ligation efficiency by removing the problematic ligation process

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

4Adaptability or versatility

If random ligation of two different sequence tags is performed using DNA ligase, then the versatility of tag attachment is improved, but the precision of di-tagging decreases due to random ligation products

Engineering Contradiction:

Improvetag attachment flexibilityVSAvoiddi-tagging accuracy

Core Design Contradiction:

Adaptability or versatilityVSManufacturing precision

Solution Approach 1:

The invention replaces the random ligation mechanism with a controlled tagmentation mechanism using Tn5 transposase. The transposase systematically inserts adapters at defined locations during fragmentation, ensuring that both ends of each DNA fragment receive sequencing adapters in a controlled manner, thereby achieving high di-tagging accuracy while maintaining versatility through the programmable nature of tagmentation

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Data Source

PatentUS8722585B2Methods of making di-tagged DNA libraries from DNA or RNA using double-tagged oligonucleotides

Publication Date: 2014.05.13 GNOMEGEN

AI summary

Disclosed are methods, compositions and kits related to making double-tagged DNA libraries from RNA/DNA samples. A double-tagged oligonucleotide (DTO) is employed to efficiently add two different tags to ends of DNAs to make a double-tagged DNA libraries. Also disclosed are methods to make mate pair libraries using the double-tagged oligonucleotide, and methods to make double-tagged single stranded DNA. The double-tagged DNA libraries of the invention are ready to be used on next generation sequencing machines.