Molecular Indexing Internal Sequences Nucleic Acid
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods of molecular barcoding and sequence analysis are limited by their focus on the 3′ end of transcripts, high sequencing error rates, low read throughput, and the absence of molecular barcoding, which restricts accurate sequence analysis and scalability.
Innovation Solution
The method involves hybridizing an oligonucleotide with a molecular barcode to a target nucleic acid, extending it to generate a circularized nucleic acid molecule with the barcode in close proximity, and amplifying it to produce amplicons for sequencing, allowing for accurate labeling and analysis of nucleic acids across their length.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of stationary object
If molecular barcoding is performed at the 3' end of transcripts using current methods, then the sequencing can be performed with standard Illumina platforms, but the analysis is limited to only the 3' end region and cannot capture upstream sequences
Solution Approach 1:
The method segments the transcript into multiple regions by performing barcoding at different positions (3' end, internal sequences, and 5' end) using different primer sets. This allows comprehensive coverage of the entire transcript length while maintaining the ability to analyze each region separately with standard sequencing platforms
Solution Approach 2:
The invention transitions from one-dimensional 3' end barcoding to multi-dimensional barcoding by adding positional dimensionality. Molecular barcodes are now attached at multiple positions along the transcript (3' end, internal regions, 5' end), enabling analysis across the full length of the nucleic acid molecule rather than being confined to a single location
2Adaptability or versatility
If gene-specific reverse transcription primers with large amounts of molecular barcodes are designed against each gene, then molecular barcoding of upstream sequences can be achieved, but the manufacturing cost becomes prohibitively expensive
Solution Approach 1:
The method employs universal barcoded primers that can bind to multiple different gene targets through the use of capture probes. Instead of designing unique barcoded primers for each gene, a single set of universal primers with molecular barcodes can be used across the entire gene pool, dramatically reducing manufacturing costs while maintaining comprehensive barcoding capability
Solution Approach 2:
The invention introduces capture probes as intermediary molecules that mediate between the universal barcoded primers and gene-specific targets. The capture probes contain gene-specific sequences that hybridize to target mRNA, while the universal primers bind to the capture probes, enabling cost-effective barcoding of any gene without requiring custom barcoded primers for each target
3Length of stationary object
If long read sequencing methods are used to sequence upstream regions, then the read length is sufficient to cover internal sequences, but the sequencing error rate is high and read throughput is low
Solution Approach 1:
The method extracts and sequences only the molecular barcode and associated target sequence regions using standard short-read Illumina sequencing. By designing the barcoding system to place the molecular barcode in close proximity to the target sequence, the system enables accurate sequencing of barcode-target pairs without requiring long-read technologies, thus maintaining high sequencing accuracy and throughput
4Ease of operation
If molecular barcodes are attached to the 3' end of transcripts, then the barcoding process is simple and scalable, but the sequence analysis is restricted to the 3' end region only
Solution Approach 1:
The transcript analysis is segmented into multiple regions (3' end, internal sequences, 5' end) with each region barcoded using appropriate primer sets. This segmentation allows the simple 3' end barcoding approach to be combined with internal and 5' end barcoding, maintaining operational simplicity while expanding analysis scope to cover the entire transcript
Solution Approach 2:
The method performs preliminary barcoding of internal sequences and 5' end regions during the same reverse transcription and amplification steps used for 3' end barcoding. By integrating multiple barcoding operations into a unified workflow, the system maintains the simplicity and scalability of the original approach while preparing samples for comprehensive multi-region analysis
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 enables high-throughput, accurate sequence analysis and quantification of nucleic acids, overcoming limitations of existing methods by allowing molecular barcoding at any position along the nucleic acid and improving scalability.
Implementation Method 1
hybridizing an oligonucleotide comprising a molecular barcode with a first nucleic acid molecule comprising the target nucleic acid
Implementation Method 2
extending the oligonucleotide to generate a second nucleic acid molecule comprising the molecular barcode and the target nucleic acid
Data Source
AI summary
The present disclosure relates to compositions, methods and kits for labeling an internal sequence of a target nucleic acid molecule with molecular barcodes. In some embodiments, the methods comprise intramolecular circulation of a labeled target nucleic acid molecule. Further provided methods for generating sequencing libraries comprising overlapping fragments covering the full length of a target nucleic acid molecule, sequencing the libraries using the methods disclosed herein, and methods of analyzing sequencing results therefrom.