Dual Indexing Nucleic Acid Barcoding for Sequencing Accuracy
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Solution Overview
Problem
Current methods for processing biological samples, such as those used in sequencing applications like ATAC-seq, face challenges with misassignment of index molecules leading to inaccurate sequencing results due to index hopping, where index tag sequences from one library are inadvertently added to nucleic acid from a different library.
Innovation Solution
The use of dual indexing and barcoding systems, including nucleic acid barcode molecules with specific primer binding and tag binding sequences, and primer molecules with sample identification sequences, to uniquely identify and distinguish between samples, thereby preventing misassignment during sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If single indexing is used for nucleic acid processing, then the system complexity is low and processing is simple, but misassignment of index molecules occurs leading to inaccurate sequencing results
Solution Approach 1:
The patent divides the single index into two separate indices (first index and second index), where each index independently identifies samples. This segmentation prevents index hopping misassignment because both indices must match correctly for accurate sample identification, thereby improving measurement precision while increasing system complexity through dual-indexing architecture
2Reliability
If dual indexing with barcode molecules and primer molecules is implemented, then sample identification accuracy improves, but the complexity of the processing system increases
Solution Approach 1:
The patent incorporates barcode sequences and sample identification sequences into the nucleic acid molecules during library preparation before sequencing. This preliminary action ensures that each nucleic acid is pre-tagged with unique identifiers, enabling accurate sample assignment during sequencing without requiring complex real-time tracking systems
Solution Approach 2:
The patent uses barcode molecules and primer molecules as intermediary elements that facilitate accurate sample identification. These intermediaries carry sample-specific sequences that bridge the connection between physical samples and sequencing reads, improving reliability while managing system complexity through standardized molecular mediators
Data Source
AI summary
While robust multiplexed high-throughput systems allow for widespread use of genome-wide chromatin accessibility (e.g., using ATAC-seq) and other next-generation sequencing studies at single cell resolution, misassignment of index molecules between samples hampers the accuracy of the sequencing result. The present disclosure provides kits, methods, and compositions for dual-indexing to allow for accurate assignment of sequencing reads to their original source samples.