Nucleic Acid Library Inversion for 5' Sequence Recovery
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Solution Overview
Problem
Sequencing nucleic acid libraries generated from single-cells or spatial array analyses often bias capture towards the 3′ end of analytes due to fragmentation and subsequent ligation of sequencing adapters, making it difficult to sequence regions more than about 1 kilobase away from the 3′ end, which limits the analysis of critical sequences such as V(D)J sequences and CDR sequences important for understanding immune cell receptor clonality.
Innovation Solution
The methods involve manipulating nucleic acid libraries by removing a portion of the sequence or reversing the orientation of sequences from double-stranded or single-stranded members, using techniques like restriction endonuclease cleavage, ligation, and amplification with specific primers to bring 5′ sequences closer to the 3′ end, enabling the sequencing of regions further away from the poly(A) tail.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If standard sequencing preparation with fragmentation and adapter ligation is used, then sequencing efficiency is improved, but capture bias towards the 3′ end occurs, making it difficult to sequence regions more than 1 kb away from the 3′ end
Solution Approach 1:
The patent applies inversion by reversing the orientation of the nucleic acid library or portions thereof. Instead of sequencing from the conventional 3′ end direction, the method inverts the library orientation to enable sequencing from the 5′ end direction, thereby recovering 5′ sequence information that would otherwise be lost due to 3′ bias in standard sequencing preparation
Solution Approach 2:
The patent applies preliminary action by performing restriction endonuclease cleavage and ligation steps before sequencing to modify the library structure. These preliminary manipulations include adding restriction sites, circularizing fragments, and performing multiple rounds of ligation to create a library configuration that enables 5′ end sequencing while maintaining sequencing efficiency
2Loss of information
If restriction endonuclease cleavage and ligation steps are added to manipulate nucleic acid libraries, then 5′ sequence capture is improved, but process complexity increases
Solution Approach 1:
The patent applies segmentation by dividing the library preparation process into distinct modular steps: initial restriction cleavage, first ligation to add adapters, optional circularization, second ligation for further modification, and amplification. Each step uses specific restriction enzymes and ligases with defined recognition sites, allowing systematic manipulation of the library to enable 5′ sequencing while maintaining process control
Solution Approach 2:
The patent uses restriction endonuclease recognition sequences and ligation intermediates as mediators between the original nucleic acid library and the final sequencing-ready product. These intermediary elements (restriction sites, adapter sequences, circularized forms) facilitate the transformation of the library structure without requiring direct manipulation of the target sequences themselves
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 identification of critical sequences beyond 1 kb from the 3′ end, enhancing the analysis of immune cell receptors like T-cell receptors and B-cell receptors, and providing high spatial resolution analyte and expression data within biological samples.
Implementation Method 1
contacting the double-stranded member of the nucleic acid library of step (a) with a first restriction endonuclease that cleaves the first restriction endonuclease recognition sequence at each end of the double-stranded member of the nucleic acid library
Implementation Method 2
ligating ends of the double-stranded member of the nucleic acid library of step (b) to generate a first double-stranded circularized nucleic acid
Implementation Method 3
amplifying the first double-stranded circularized nucleic acid using a first and a second primer to generate a first double-stranded nucleic acid product
Data Source
AI summary
Provided herein are methods, compositions, and kits for removing a portion of a sequence in a member of a nucleic acid library.