sgRNA Sequencing Linker with UMI Loop Structure for Bias Reduction
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Solution Overview
Problem
Current sgRNA sequencing technologies face challenges in constructing libraries with high yield and accuracy due to chemical modifications, leading to PCR amplification bias and linking bias, which affect the representation of RNA abundance and sequence information.
Innovation Solution
A novel linker system comprising a 3′ linker and a 5′ linker with specific non-random and random sections, forming a neck ring structure and loop-forming DNA sections, is used to construct sgRNA sequencing libraries, allowing for reverse transcription without additional primers and reducing bias through unique molecule identifiers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If chemical modifications are performed at the 5'-end and 3'-end of sgRNA to improve storage stability, then storage stability is improved, but library construction difficulty increases and library yield decreases
Solution Approach 1:
The patent introduces a linker molecule as an intermediary between the modified sgRNA and the sequencing platform. The linker contains a modification-resistant section that can tolerate chemical modifications at the sgRNA ends while still enabling successful library construction and sequencing, thus mediating between the stable but difficult-to-process modified RNA and the sequencing requirements
2Stability of the object's composition
If chemical modifications are performed at the 5'-end and 3'-end of sgRNA to improve storage stability, then storage stability is improved, but library yield decreases
Solution Approach 1:
The linker serves as a mediator that protects the modified sgRNA ends from interfering with library construction efficiency. By providing a buffer zone with modification-resistant sequences, the linker enables high-yield library construction from chemically modified sgRNA that would otherwise be difficult to process
3Quantity of substance
If PCR amplification is performed to amplify the sgRNA library, then sequencing material is sufficient, but PCR amplification bias occurs causing inaccurate RNA abundance representation
Solution Approach 1:
The patent incorporates unique molecule identifiers (UMIs) into the linker sequences before PCR amplification. These pre-inserted unique sequences allow for bioinformatic correction of PCR amplification bias during data analysis, enabling accurate RNA abundance measurement even after necessary PCR amplification to generate sufficient sequencing material
4Ease of manufacture
If a conventional linker is used for sgRNA sequencing library construction, then library construction can proceed, but linking bias occurs affecting sequencing accuracy
Solution Approach 1:
The patent designs the linker with distinct functional regions having different properties: a modification-resistant section with specific sequence characteristics to minimize linking bias, a random section for UMI functionality, and standardized sections for adapter annealing. This local differentiation of linker properties enables both efficient library construction and accurate sequencing by addressing different functional requirements in different regions
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
The linker system enhances library construction efficiency, reduces background noise, and accurately reflects RNA abundance, improving the sequencing results by minimizing PCR amplification bias and linker dimer contamination.
Implementation Method 1
the second non-random section is reversely complementary to the third non-random section so as to form a neck ring structure in conjuncture with the loop-forming DNA section
Implementation Method 2
the loop-forming DNA section is composed of a first loop-forming section and a second loop-forming section from the 5'-end to the 3'-end
Implementation Method 3
the third non-random section is used as a primer for sgRNA reverse transcription and replication
Data Source
AI summary
The present invention relates to the technical field of molecular biology, and in particular, to a sgRNA sequencing linker and use thereof. The sgRNA sequencing 3′ linker sequentially comprises the following sections from a 5′-end to a 3′-end: a first non-random section, a first random section, a second non-random section, a loop-forming DNA section, and a third non-random section, wherein the first non-random section is used for being linked to 3′-end of the sgRNA; the first random section comprises 3 to 12 basic groups; the second non-random section is reversely complementary to the third non-random section so as to form a neck ring structure in conjuncture with the loop-forming DNA section; the third non-random section is used as a primer for sgRNA reverse transcription and replication; the loop-forming DNA section is composed of a first loop-forming section and a second loop-forming section from 5′-end to the 3′-end; the third non-random section and the second loop-forming section can be combined with the first sequencing linker primer sequence in a complementary pairing mode.