Single-Cell Transcript Enrichment for Barcode-Linked Genotyping
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Solution Overview
Problem
Existing high-throughput single-cell RNA-seq technologies face challenges in sequencing defined portions of transcripts, particularly those at the 5′ end, while maintaining cell identification, and struggle to efficiently distinguish tumor cells from normal cells and differentiate minority cell populations.
Innovation Solution
A method is developed to construct a library of enriched single cell RNA transcripts with a cell barcode in close proximity to the desired transcript sequence, using targeted capture and PCR amplification to enrich for specific gene regions, followed by sequencing to distinguish cells by genotype.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If random sequencing is used on standard 3'-barcoded libraries, then single cell identification is maintained, but sequencing efficiency for defined portions of transcripts is poor
Solution Approach 1:
The patent applies preliminary action by performing targeted PCR enrichment before sequencing to pre-concentrate the desired transcript regions (such as 5' ends and variable regions) that are of interest. This preliminary enrichment step ensures that when sequencing occurs, the desired sequences are already in close proximity to their barcodes and significantly enriched, thereby improving both sequencing efficiency and measurement precision without requiring changes to the fundamental barcoding approach
Solution Approach 2:
The patent extracts and enriches specific portions of transcripts (particularly 5' ends and variable regions of TCR/BCR) from the full-length cDNA molecules. By using targeted PCR with primers designed to amplify only the regions of interest, the method separates the desired sequences from the rest of the transcript, bringing them into close proximity with their associated barcodes for efficient sequencing
2Adaptability or versatility
If 3'-barcoded libraries are used, then cell barcodes are attached to all transcripts, but transcripts greater than 1 kb away from the barcode cannot be efficiently sequenced
Solution Approach 1:
The patent introduces targeted PCR enrichment as an intermediary step between library construction and sequencing. This intermediary process uses gene-specific primers to amplify and bring distant transcript regions (greater than 1 kb from the 3' barcode) into close proximity with the barcode, enabling efficient sequencing of previously inaccessible regions while maintaining the versatility of 3'-barcoded libraries
Solution Approach 2:
By performing targeted enrichment through PCR before sequencing, the method preliminarily concentrates the desired distant transcript regions adjacent to their barcodes. This preliminary action transforms the library composition to favor sequences that are otherwise too distant for efficient random sampling, thereby expanding transcript region coverage without sacrificing data acquisition efficiency
3Measurement precision
If standard single-cell RNA-seq techniques are used, then unbiased detection of cell types is achieved, but minority cell populations and tumor cells cannot be distinguished
Solution Approach 1:
The patent extracts and enriches specific transcript regions (such as variable regions of TCR/BCR and other cell-type-specific markers) that serve as discriminatory features. By focusing sequencing efforts on these extracted, enriched regions rather than random sequencing of all transcripts, the method achieves high measurement precision for cell type identification and enables detection of minority populations including tumor cells with greater sensitivity
4Productivity
If targeted sequencing of specific transcript regions is attempted, then sequencing efficiency improves, but cell barcode maintenance becomes difficult
Solution Approach 1:
The patent performs targeted PCR enrichment with primers designed to amplify the desired transcript regions while simultaneously incorporating or preserving the cell barcode in the amplification product. This preliminary enrichment step ensures that the barcode is maintained and brought into close proximity with the targeted sequence, achieving both high sequencing efficiency and reliable barcode retention
Solution Approach 2:
The targeted PCR process creates copies of the desired transcript regions that include the cell barcode. By designing primers that amplify both the target sequence and the adjacent barcode region, the method produces enriched copies where the barcode is reliably attached to the targeted sequence, maintaining reliability while improving sequencing efficiency
Data Source
AI summary
The present invention relates to methods of detecting region(s) of interest in a gene comprising a polyA tail. The region(s) of interest can include gene(s), region(s), mutation(s), deletion(s), insertion(s), indel(s), and/or translocation(s). The region(s) can be greater than or less than 1 kilobases from the polyA tail. Methods can include forming a library of single cell transcripts comprising the region(s) in close proximity to a cell barcode and a unique molecular identifier (UMI). Methods for distinguishing cells by genotype can include amplifying the transcripts using PCR methods and detecting the cell barcode and UMI using single cell sequencing methods. Transcripts can be enriched using tagged region-specific PCR primers. Cell barcodes can be brought into close proximity to the region(s) by circularizing the transcripts. Sequencing of the transcripts can include using primer binding sites added during PCR amplification and library indexes for multiplexed sequencing.