Bead-Linked Transposomes for Strand-Specific RNA Sequencing
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Solution Overview
Problem
Current RNA sequencing methods face challenges in preparing strand-specific libraries, particularly with tagmentation techniques that result in 3' bias and inefficiencies due to asymmetrical transposome usage, and lack methods for simultaneous RNA and DNA sequencing from a single sample without significant sample loss or complexity.
Innovation Solution
The method involves immobilizing transposome complexes on a solid support, capturing RNA with polyT oligonucleotides, synthesizing cDNA, and performing tagmentation to generate 5'-tagged DNA:RNA duplexes, allowing for symmetrical tagging and reducing 3' bias, while also enabling simultaneous RNA and DNA sequencing by segregating biomolecules on different beads.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If asymmetrical tagmentation with bead-linked transposomes is used, then library preparation efficiency is improved, but 3' bias is introduced and strand-specific information is lost
Solution Approach 1:
The patent applies asymmetry in reverse - it uses symmetrical transposome complexes (both A14 and B15 transposases) to achieve symmetrical tagging of both ends of DNA fragments. This symmetry allows retention of strand-specific information while maintaining the efficiency benefits of tagmentation. The transposomes are designed with complementary strand specificity, enabling them to tag both strands equally without introducing 3' bias.
Solution Approach 2:
Instead of using asymmetrical transposomes that tag only one end (as in prior art), the patent inverts the approach by using transposomes that can tag both ends symmetrically. This inversion allows the method to achieve strand-specific RNA sequencing while maintaining high efficiency, effectively solving the contradiction by reversing the conventional asymmetrical approach.
2Adaptability or versatility
If RNA is converted to double-stranded cDNA format prior to sequencing, then sequencing compatibility is improved, but strand-specific information is lost
Solution Approach 1:
The patent performs tagmentation during the cDNA synthesis process itself, before the second strand is fully synthesized. This preliminary tagging action occurs when the RNA template is still present and can guide the transposomes to specific locations, thereby preserving strand-specific information. The tagging is done in advance, during the first-strand synthesis, rather than after complete double-stranded cDNA formation.
3Measurement precision
If unique molecular identifiers are attached at the 3' end for single-cell RNA sequencing, then quantitative measurement is improved, but isoform expression analysis is limited
Solution Approach 1:
The patent creates a universal method that can simultaneously achieve quantitative measurement (through UMI tagging) and isoform expression analysis (through full-length transcript coverage). The symmetrical tagmentation approach allows UMIs to be placed at both ends of fragments, enabling both 3' quantitative analysis and full-length isoform characterization from the same library, thus making the method multi-functional.
Solution Approach 2:
The patent adds a new dimension to UMI placement by positioning UMIs at both the 3' and 5' ends of cDNA fragments through symmetrical tagmentation. This dimensional change from single-end to dual-end UMI placement enables simultaneous quantification and isoform analysis, transforming the limitation into a capability for comprehensive transcript characterization.
4Productivity
If sample is split for separate RNA and DNA sequencing, then sequencing depth is improved, but sample loss and workflow complexity increase
Solution Approach 1:
The patent merges RNA and DNA sequencing workflows into a single integrated process. By using symmetrical tagmentation that works on both RNA and DNA samples with the same protocol, the method eliminates the need to split samples into separate preparation workflows. Both RNA and DNA can be processed simultaneously from a single sample, maintaining sequencing depth while reducing complexity.
Solution Approach 2:
The patent creates a universal library preparation method that handles both RNA and DNA sequencing with the same protocol and reagents. The symmetrical transposome system is universally applicable to different nucleic acid types, allowing a single sample to be used for both RNA and DNA sequencing without requiring separate workflows, thereby reducing complexity while maintaining productivity.
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 enhances library preparation efficiency by reducing 3' bias and allows for comprehensive RNA and DNA sequencing from a single sample, improving yield and accuracy in RNA sequencing and enabling multi-omic analyses without sample splitting.
Implementation Method 1
the Tn5 enzyme catalyzes the translocation of adapters required for sequencing to the ends of double-stranded DNA or cDNA through a 'cut-and-paste' mechanism referred as tagmentation
Implementation Method 2
adding a reverse transcriptase polymerase under conditions to synthesize cDNA and generate immobilized DNA:RNA duplexes on the capture oligonucleotides
Implementation Method 3
wherein the sample is applied to the solid support under conditions wherein the 3' end of the target RNA binds to the capture oligonucleotides
Data Source
AI summary
This application describes methods of preparing an immobilized library of tagged RNA fragments. Also described herein are a number of methods of preparing DNA and RNA sequencing libraries from a single sample. These methods can include library preparation from single cells.