Capture Oligonucleotide for Full-Length mRNA Sequencing
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Solution Overview
Problem
Current next-generation sequencing platforms cannot efficiently capture full-length mRNAs inclusive of their poly-A tails due to limitations in handling homopolymeric sequences longer than 30 nucleotides, leading to the discard of poly(A) sequences during library preparation and analysis, which hampers the detailed composition study of transcriptomes.
Innovation Solution
A single-pot protocol using a system comprising a single-stranded capture oligonucleotide with a non-extendable end, a capture sequence, a selectively cleavable base, barcode sequences, and a terminal adapter, along with enzymes like deoxyuracil glycosylase and reverse transcriptase, enables the efficient end-to-end capture of mRNAs as cDNA, including their poly-A tails, in a single reaction volume.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard base-calling algorithms are used in NGS platforms, then sequencing can be performed, but homopolymeric sequences longer than 30 nucleotides cannot be handled, leading to loss of poly(A) tail information
Solution Approach 1:
The invention extracts the poly(A) tail capture function from the standard NGS workflow by using oligo-dT primers that specifically bind to poly(A) tails, separating this function from the general sequencing process. This allows poly(A) inclusive sequencing to be performed with standard NGS platforms without requiring modification of the base-calling algorithms themselves.
Solution Approach 2:
The invention performs preliminary enrichment of poly(A) containing RNAs using oligo-dT primers before sequencing. This preliminary action captures the poly(A) tails in the cDNA synthesis step, ensuring they are present in the final library even though the sequencing platform itself cannot natively handle homopolymeric sequences longer than 30 nucleotides.
2Productivity
If poly(A) sequences are discarded during library preparation, then homopolymeric sequence limitations are avoided, but full-length mRNA capture is compromised
Solution Approach 1:
The invention makes the oligo-dT primer multi-functional: it serves both as a capture element for poly(A) tails and as a primer for cDNA synthesis. The primer contains a poly(dT) region for binding poly(A) tails and additional sequence elements for priming reverse transcription, allowing simultaneous capture and full-length cDNA synthesis in a single step.
Solution Approach 2:
The invention nests the poly(A) tail capture function within the cDNA synthesis process. The oligo-dT primer is designed with nested functional regions: the poly(dT) portion binds the poly(A) tail while the 5' portion provides priming capability, effectively nesting the capture function inside the synthesis reaction.
3Measurement precision
If multi-step protocols are used for full-length mRNA capture, then poly(A) inclusive sequencing is achieved, but protocol complexity increases and droplet-based single-cell RNA sequencing becomes difficult
Solution Approach 1:
The invention merges multiple functions into a single oligo-dT primer design: poly(A) tail binding, cDNA priming, and full-length transcript capture are all achieved with one primer in a single reaction step. This eliminates the need for separate enrichment and synthesis steps required by previous multi-step protocols like FLAM-seq and PAISO-seq.
Solution Approach 2:
The oligo-dT primer is designed as a universal reagent that works across different sequencing applications including bulk RNA-seq and droplet-based single-cell RNA sequencing. The primer's design allows it to function in both simple tube-based reactions and complex microfluidic droplet systems without modification.
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 allows for the biologically informative capture of full-length mRNAs, preventing artifactual truncation and providing isoform-level information, enabling high-resolution inference of RNA velocity and poly-A length as a temporal expression proxy, suitable for droplet-based single-cell RNA sequencing.
Implementation Method 1
an enzyme or combination of enzymes capable of cleaving the selectively cleavable base only in a DNA: DNA duplex or DNA/RNA heteroduplex
Implementation Method 2
a reverse transcriptase (RT)
Implementation Method 3
a capture sequence, wherein the capture sequence is an oligo-dT sequence
Data Source
AI summary
Methods and compositions for a single- or multi-pot protocol for the efficient end to end capture of RNAs (inclusive of their poly-A tail or their 3′ end) is described. Capture oligonucleotides containing a 3′ non-extendable end and a selectively cleavable base upstream of an oligo-dT or oligo-dN and a 5′ sequence containing unique molecular identifiers, and 2) a deoxyuracil glycosylase that acts only on a deoxyuracil present in a DNA: DNA duplex or DNA/RNA heteroduplex are used. A dual template switching mechanism may be used.