Polynucleotide Library Sequencing for Single-Cell Transcriptome Analysis
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Solution Overview
Problem
Current methods for single-cell transcriptome sequencing are limited in throughput and unable to capture full-length immune receptor sequences, making it difficult to analyze gene expression and immune repertoire effectively.
Innovation Solution
The method involves producing a polynucleotide library from individual cells by adding adaptors to barcoded single-stranded polynucleotides, allowing for the generation of full-length target sequences and comprehensive transcriptome analysis, with each cell tagged with unique molecular and vessel barcodes for high-throughput sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If existing single-cell transcriptome sequencing methods are used, then gene expression analysis can be performed, but throughput is limited and full-length immune receptor sequences cannot be captured
Solution Approach 1:
The method segments the sequencing process into distinct stages: (1) cell lysis and reverse transcription with template switching to capture full-length cDNA, (2) barcode attachment to link transcripts to parent cells, (3) amplification of both target and transcriptome sequences, and (4) high-throughput sequencing. This segmentation allows simultaneous capture of full-length immune receptor sequences and comprehensive transcriptome data across many cells, resolving the contradiction between throughput and capture completeness
Solution Approach 2:
The method performs preliminary actions by: (1) using template switching oligos with barcodes during reverse transcription to pre-tag full-length cDNA molecules with cell-specific identifiers, (2) performing targeted amplification of immune receptor sequences before transcriptome amplification to ensure complete capture, and (3) preparing adaptor-ligated libraries in advance for high-throughput sequencing. These preliminary actions enable subsequent high-throughput processing while maintaining complete sequence capture
2Measurement precision
If barcoding is used to track single cells, then cell-specific transcriptome analysis is enabled, but the complexity of the library preparation process increases
Solution Approach 1:
The method merges multiple functions into unified steps: (1) template switching oligos simultaneously serve as reverse transcription primers and barcode carriers, (2) the same barcode system is used for both target sequence identification and transcriptome attribution, and (3) adaptor ligation combines library preparation with sequencing primer attachment. This merging reduces the number of separate operations needed while maintaining precise cell-specific tracking capability
Solution Approach 2:
The barcode system is designed with universal applicability: (1) the same barcode structure is used across different cell types and experimental conditions, (2) barcodes function both as cell identifiers and as handles for amplification and sequencing, and (3) the adaptor design is universal for both target and transcriptome sequences. This multi-functionality simplifies the overall process while maintaining measurement precision
3Manufacturing precision
If full-length target sequences are captured, then immune repertoire analysis is improved, but the amount of sequencing data and processing requirements increase
Solution Approach 1:
The method extracts and prioritizes full-length target sequences through: (1) template switching that specifically captures complete cDNA molecules during reverse transcription, (2) targeted amplification steps that enrich for immune receptor sequences before transcriptome amplification, and (3) adaptor ligation that prepares these extracted sequences for efficient sequencing. This extraction approach focuses sequencing capacity on obtaining complete target sequences rather than attempting to sequence all transcripts at equal depth
Data Source
AI summary
Provided herein are methods for target gene sequencing and single cell barcoding in conjunction with analysis of gene expression in single cells. In some embodiments, the target gene is an immune molecule, such as an antibody or TCR. In some embodiments, the methods can be used to carry out transcriptome sequencing, e.g., RNA sequencing, to capture transcriptome of single cells paired with full receptor immune receptor sequences such that information about the immune repertoire and transcriptome of a cell can be determined. Also provided are polynucleotide libraries for use in carrying out transcriptome analysis and immune molecule, e.g., antibody or TCR, sequencing.