Dual-Index Nucleic Acid Fragments for Accurate Read Assignment
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Solution Overview
Problem
Existing genetic sequencing techniques face challenges with accuracy due to errors and noise in multi-sample preparations, leading to index cross-contamination and misassignment of sequencing reads, particularly in low-plexity runs.
Innovation Solution
A dual-indexing strategy is employed, where each nucleic acid fragment is labeled with a plurality of unique indices at multiple sites, ensuring diverse index sequences to improve sequencing accuracy by reducing misassignment and index hopping.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional single-indexing or combinatorial indexing is used in multi-sample sequencing, then throughput is improved, but index cross-contamination and misassignment errors increase
Solution Approach 1:
The patent divides the indexing system into two independent index sites (first index and second index) on opposite strands of the nucleic acid fragment. Each site uses a separate index sequence from its own pool, creating segmented indexing that prevents cross-contamination between samples while maintaining high throughput multiplexing capability
Solution Approach 2:
The patent creates a composite indexing system by combining two distinct index sequences (one from the first index pool and one from the second index pool) on each nucleic acid fragment. This composite dual-indexing approach provides redundant sample identification that eliminates index hopping errors while preserving multiplexing efficiency
2Productivity
If combinatorial indexing approach is used to increase sample multiplexing, then productivity increases, but measurement precision of sample origin decreases due to index hopping
Solution Approach 1:
The patent segments the indexing function into two independent sites located on opposite strands of the nucleic acid fragment. Each site independently identifies the sample origin using sequences from separate pools, preventing index hopping between samples while maintaining high multiplexing capacity through the combination of both indexes
3Device complexity
If single indexing per fragment is used, then device complexity is reduced, but reliability of sample association decreases due to errors and noise
Solution Approach 1:
The patent employs a composite dual-indexing system where each nucleic acid fragment carries two independent index sequences from separate pools. This composite approach provides redundant sample identification that corrects errors and noise in the sequencing data, significantly improving sample association reliability while maintaining manageable system complexity through standardized protocols
Data Source
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AI summary
Presented herein are techniques for indexing of nucleic acid, e.g., for use in conjunction with sequencing. The techniques include generating indexed nucleic acid fragments from an individual sample, whereby the index sequence incorporated into each index site of the nucleic acid fragment is selected from a plurality of distinguishable of index sequences and such that the population of generated nucleic acid fragments represents each index sequence from the plurality. In this manner, the generated indexed nucleic acid fragments from a single sample are indexed with a diverse mix of index sequences that reduce misassignment due to index read errors associated with low sequence diversity.