Antibody Expression Constructs with UMIs for Sequencing Depth
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Solution Overview
Problem
Current methods for high throughput sequencing of antibody protein products are limited by the size of the expression constructs, which restricts the sequencing depth and efficiency, especially when using standard constructs and sequence methods.
Innovation Solution
The development of polynucleotide expression constructs that include a universal molecular identification (UMI) barcode and a molecular barcode, such as an optical barcode, along with a nucleotide sequence specific for a universal reverse transcriptase (RT) primer, facilitates high throughput sequencing by enabling the identification of molecule sequences using optical fluidic devices.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard antibody expression constructs are used for sequencing, then the sequencing process is simpler, but only the ends of the polynucleotide molecule are sequenced and the entire molecule cannot be sequenced unless exported one bead at a time
Solution Approach 1:
The expression construct is segmented into distinct functional modules: a molecular barcode region for identification, a UMI region for unique molecule tagging, the antibody coding sequence, and a universal RT primer binding site. This segmentation allows the sequencing system to target and sequence specific regions efficiently, enabling complete molecule sequencing while maintaining high throughput.
Solution Approach 2:
The patent introduces molecular barcodes and UMIs as intermediary elements that bridge the antibody coding sequence and the sequencing detection system. These intermediaries enable the sequencing apparatus to identify, tag, and track individual antibody molecules, facilitating complete sequencing of the polynucleotide molecule while maintaining high throughput capabilities.
2Measurement precision
If optical barcodes are used for high throughput sequencing, then single cell screening capability is enhanced, but the size of the expression construct limits the sequencing depth
Solution Approach 1:
The patent adds informational dimensions to the expression construct by incorporating molecular barcodes and UMIs. These additional nucleotide sequences provide multiple layers of identification (optical barcode for spatial positioning, UMI for molecule uniqueness) that enable high-resolution single cell screening without proportionally increasing the physical constraints on sequencing depth.
Solution Approach 2:
The molecular barcodes and UMIs are incorporated into the expression construct during the cloning stage, before sequencing. This preliminary tagging of each antibody molecule with unique identifiers allows the sequencing system to efficiently track and sequence complete molecules later, overcoming the limitation of construct size by pre-organizing the information architecture.
3Measurement precision
If the entire polynucleotide molecule is sequenced by exporting one bead at a time, then complete sequencing is achieved, but the sequencing throughput is significantly reduced
Solution Approach 1:
The universal RT primer binding site serves multiple functions: it enables reverse transcription of the antibody coding sequence, provides a consistent entry point for sequencing initiation, and works with the molecular barcode and UMI systems. This multi-functionality allows the sequencing system to process complete molecules efficiently without requiring separate procedures for different regions, thus maintaining high throughput while achieving complete sequencing.
Data Source
AI summary
The disclosure provides for a polynucleotide molecule encoding an antibody protein product, wherein the polynucleotide molecule comprises i) a nucleotide sequence specific for the addition of a molecular barcode by a template switching reverse transcriptase (RT) and a unique molecular identifier (UMI) barcode, and a nucleotide sequence specific for a universal RT primer to facilitate high throughput sequencing.