Single-Cell Transcriptome Barcoding Without Cell Isolation
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Solution Overview
Problem
Existing methods for whole transcriptome analysis in single cells require physical isolation of cells and are inefficient for detecting rare nucleic acid targets, necessitating a more robust method for barcoding and detection.
Innovation Solution
A method utilizing a split-pool process with reverse transcriptase to assemble compound barcodes on nucleic acid targets in individual cells, employing oligonucleotide primers and barcode subunits, and nucleic acid polymerases with terminal transferase activity to extend copy strands with non-templated nucleotides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If ligation is used to assemble compound barcodes on nucleic acid targets, then barcodes can be assembled, but the process is less efficient and requires additional reagents and reaction conditions
Solution Approach 1:
The patent replaces the mechanical ligation process with a biochemical primer extension process using reverse transcriptase. Instead of using ligase enzymes to join barcode subunits, the invention uses reverse transcriptase to synthesize complementary DNA strands that incorporate barcode subunits through nucleotide addition, eliminating the need for ligation and its associated complexity
Solution Approach 2:
The patent changes the reaction parameters from ligation conditions to reverse transcription conditions. By using reverse transcriptase with terminal transferase activity, the system operates under different biochemical parameters that enable direct synthesis of barcoded products without the intermediate ligation step, improving efficiency and reducing reagent requirements
2Measurement precision
If physical isolation of individual cells is performed, then single cell analysis can be conducted, but the process becomes more complex and time-consuming
Solution Approach 1:
The patent applies segmentation by dividing the cell population into individual reaction compartments using droplet microfluidics. Each droplet contains a single cell and undergoes independent reverse transcription and barcode assembly, enabling single-cell resolution without manual isolation. The segmented processing occurs in parallel across millions of droplets, maintaining precision while reducing time
Solution Approach 2:
The patent performs preliminary actions by pre-assembling barcode subunits and preparing reverse transcriptase reactions before cell introduction. The system pre-establishes the biochemical conditions and barcode libraries, so that when cells are processed through the droplet system, the barcoding can occur immediately without waiting for isolation and preparation steps
3Reliability
If conventional barcoding methods are used for rare nucleic acid targets, then detection can be performed, but the efficiency is insufficient for whole transcriptome analysis
Solution Approach 1:
The patent creates a universal reverse transcription-primer extension system that can detect any nucleic acid target through a single unified process. The reverse transcriptase enzyme and barcode assembly mechanism work universally across different target types (mRNA, lncRNA, viral RNA), eliminating the need for target-specific optimization and enabling efficient whole transcriptome analysis while maintaining reliable detection of rare targets
Solution Approach 2:
The patent introduces reverse transcriptase as an intermediary enzyme that mediates between the nucleic acid target and the barcode assembly process. This intermediary converts various RNA targets into complementary DNA strands while simultaneously incorporating barcodes, bridging the gap between target detection and data encoding in a single efficient step that works for both abundant and rare targets
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
Enables efficient and robust detection of multiple nucleic acid targets in individual cells without the need for cell isolation, improving the efficiency of whole transcriptome analysis.
Implementation Method 1
A unique property of the reverse transcriptase enzyme is employed to copy the barcode subunits and assemble a compound barcode
Implementation Method 2
nucleic acid polymerase having a terminal transferase activity and extending the oligonucleotide primer to form a copy strand having one or more non-templated nucleotides at the 3′-end
Data Source
AI summary
The invention is a method of single cell transcriptome analysis. The method comprises detecting multiple transcripts in each individual cell of the plurality of cells by barcoding the transcripts with a cell-specific compound barcode formed using a DNA polymerase and a terminal transferase, optionally in a single enzyme such as a reverse transcriptase.