Single-Cell Tagmentation for 3′ End Transcript Library Prep
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Solution Overview
Problem
Existing single cell nucleic acid sequencing methods require full transcript processing, which is inefficient and resource-intensive, especially when only a fraction, such as the 3′ end, needs to be sequenced.
Innovation Solution
A method involving tagmentation to insert a sequencing primer sequence into single cell-barcoded nucleic acid analytes, using a transposase to generate barcoded nucleic acid fragments suitable for library preparation, bypassing full cDNA amplification and fragmentation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If full transcript processing is performed in existing single cell sequencing methods, then complete sequence information is obtained, but processing time and reagent usage increase significantly
Solution Approach 1:
The patent extracts and processes only the necessary 3' end portion of transcripts rather than full-length transcripts. The tagmentation step specifically targets and processes only the required fraction of the transcript, eliminating unnecessary processing of the remaining portions and thereby reducing processing time while maintaining essential sequence information.
Solution Approach 2:
The patent applies partial action by processing only the 3' end region of transcripts that is sufficient for the sequencing application. This partial processing approach avoids the time-consuming full transcript processing while obtaining adequate sequence information for the intended purpose.
2Quantity of substance
If full cDNA amplification and fragmentation is performed in existing workflows, then sufficient material for sequencing is obtained, but reagent consumption and process complexity increase
Solution Approach 1:
The patent performs preliminary tagmentation on the barcoded nucleic acid molecules before full amplification. By fragmenting and adding sequencing adapters in advance through tagmentation, the workflow eliminates the need for subsequent fragmentation steps and reduces the number of amplification cycles required, thereby reducing reagent consumption and simplifying the overall process.
Solution Approach 2:
The patent merges the fragmentation and adapter addition steps into a single tagmentation operation. This combined approach replaces the traditional separate fragmentation and end-repair/adapter-ligation steps, reducing workflow complexity and reagent usage while achieving the same functional outcome.
3Reliability
If traditional library construction steps are performed, then sequencing-ready libraries are generated, but the process requires multiple steps increasing reagent usage and time
Solution Approach 1:
The patent uses tagmentation as an intermediary step that simultaneously achieves fragmentation and sequencing adapter incorporation. This mediator process replaces multiple traditional library construction steps (fragmentation, end-repair, adapter ligation) with a single enzymatic reaction, improving productivity while maintaining library construction reliability through the use of well-established transposase chemistry.
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
Streamlines library construction and reduces reagent usage by directly generating barcoded nucleic acid fragments for sequencing, enhancing the efficiency of single cell transcriptomic analysis.
Implementation Method 1
using a transposase to insert a sequencing primer sequence into single cell-barcoded nucleic acid analytes, or an intermediate or complement thereof, to thereby generate single cell-barcoded nucleic acid fragments
Implementation Method 2
hybridizing the mRNA molecule to a barcode nucleic acid molecule attached to a solid support, wherein the barcode nucleic acid molecule comprises: (i) a barcode sequence and (ii) a domain that hybridizes to a 3' sequence of the mRNA molecule
Implementation Method 3
extending the barcode nucleic acid molecule, thereby generating a barcoded nucleic acid molecule annealed to the mRNA molecule, wherein the barcoded nucleic acid molecule comprises a sequence complementary to the mRNA molecule
Implementation Method 4
the solid support is magnetic, and the separating comprises magnetically separating
Implementation Method 5
the barcode nucleic acid molecule comprises an affinity tag, and the separating comprises affinity purification
Data Source
AI summary
Provided herein are methods, systems, and kits for detecting analytes of interest from biological samples using tagmentation. The methods include tagmenting DNA/RNA hybrids that include an mRNA molecule and a barcode nucleic acid molecule to generate a plurality of tagmented fragments, and separating a first tagmented fragment including an end portion of the mRNA and a barcode from other tagmented fragments.