Digestible Primers for Single-Cell RNA Sequencing
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Solution Overview
Problem
High throughput single-cell RNA sequencing is hindered by primer byproducts and mispriming during reverse transcription, leading to erroneous sequence reads and inaccurate cell characterization due to the presence of primer byproducts and misprimed nucleic acids.
Innovation Solution
The implementation of digestible primers that participate in reverse transcription but are subsequently digested, preventing their involvement in nucleic acid amplification, thereby reducing primer byproducts and mispriming errors. These primers can include ribonucleotide or uracil nucleobases and are digested using enzymes like RNase or uracil-DNA glycosylase before amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If reverse transcription primers are used for RNA sequencing, then RNA can be converted to cDNA, but primer byproducts and mispriming occur leading to erroneous sequence reads
Solution Approach 1:
The patent extracts and removes the harmful primer byproducts from the system by using digestible primers that can be selectively degraded by RNase or UDG enzymes after serving their reverse transcription function, thereby eliminating the source of erroneous sequence reads while preserving the useful cDNA conversion process
Solution Approach 2:
The patent changes the chemical composition parameters of the primers by incorporating ribonucleotides or uracil residues into otherwise DNA primers, making them susceptible to enzymatic digestion while maintaining their ability to prime reverse transcription, thus resolving the contradiction between functionality and harmful byproduct generation
2Reliability
If digestible primers are used in reverse transcription, then primer byproducts are reduced, but additional digestion steps are required in the workflow
Solution Approach 1:
The patent merges the primer digestion step with existing workflow stages by performing enzymatic digestion during or after the reverse transcription reaction, rather than as a separate additional step, thereby reducing overall workflow complexity while maintaining improved accuracy
Solution Approach 2:
The patent introduces enzymatic digestion as an intermediary step that bridges the reverse transcription and amplification processes, using RNase or UDG enzymes to selectively remove digestible primers before they can interfere with subsequent amplification, thus improving accuracy without significantly increasing workflow complexity
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 significantly improves sequence read metrics, including increased percentages of mapped reads and reads with valid cell barcodes, enhancing the accuracy of single-cell analysis by eliminating primer-related errors.
Implementation Method 1
digesting the digestible primer comprises exposing the digestible primer to a RNase or uracil-DNA glycosylase
Implementation Method 2
digesting the digestible primer comprises exposing the digestible primer to a RNase or uracil-DNA glycosylase
Data Source
AI summary
Digestible primers are incorporated into single cell analysis workflows to reduce and/or eliminate primer byproducts and misprimed nucleic acids. Specifically, digestible primers can participate in a first reaction, such as reverse transcription of RNA transcripts to generate cDNA, but digestible primers are digested to prevent them from participating in subsequent reactions, such as nucleic acid amplification. For example, digestible primers can include a primer with one or more ribonucleotide nucleobases, a primer with uracil bases, a primer with deoxyuridine sequences, or a primer with ribouridine sequences. Such primers can then be digested (e.g., enzymatically digested) to remove them from interfering in subsequent nucleic acid amplification reactions.