Dual Nucleic Acid Indexing for Accurate Low-Diversity Sequencing
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Solution Overview
Problem
Existing genetic sequencing techniques face challenges with high throughput sequencing in multi-sample preparations due to errors and noise from sample defects, preparation errors, and sequencing bias, leading to inaccurate sample association and index hopping, especially in low-plexity sequencing runs.
Innovation Solution
A dual-indexing strategy is employed, where each nucleic acid fragment is labeled with a plurality of unique indices at two index sites, using a pre-set pool of index sequences to improve sequencing accuracy and reduce index hopping, ensuring each sample is associated with a distinct set of index sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional single indexing or combinatorial indexing is used in multi-sample preparations, then sequencing throughput is improved, but sample attribution accuracy deteriorates due to index cross-contamination and misassignment
Solution Approach 1:
The patent divides the indexing system into two separate index positions (first index and second index) on each nucleic acid fragment. Each position receives a distinct index sequence, creating segmented identification markers that reduce cross-contamination. This segmentation allows independent tracking of each index position, improving sample attribution accuracy while maintaining multi-sample throughput capability.
Solution Approach 2:
The patent transitions from single-dimension indexing (one index per sample) to two-dimension indexing (first index and second index per fragment). By adding another dimension of indexing, the system increases the uniqueness and specificity of sample identification, reducing misassignment errors while preserving high throughput sequencing capability.
2Measurement precision
If dual indexing with diverse unique index sequences is implemented, then sample attribution accuracy is improved, but indexing complexity increases
Solution Approach 1:
The patent employs universal adapter sequences that can accommodate multiple different index sequences at each index position. These universal adapters serve multiple functions: they provide the indexing capability, enable pool preparation from multiple samples, and work with standard sequencing workflows. This multi-functionality reduces the need for sample-specific customization, managing complexity while maintaining high attribution accuracy.
Solution Approach 2:
The patent changes the parameter of index sequence diversity by using a defined set of unique index sequences assigned to different samples. Rather than creating entirely custom indexes for each sample, the system varies the index sequence parameters within a standardized framework, reducing complexity while preserving the ability to accurately distinguish between samples.
3Productivity
If index sequences are assigned to multiple samples (combinatorial approach), then indexing efficiency is improved, but index cross-contamination increases
Solution Approach 1:
The patent segments the index assignment by dedicating specific index sequences to specific samples at each index position, rather than using combinatorial sharing. Each sample receives unique index combinations, preventing cross-contamination while maintaining efficient pool preparation. This segmentation eliminates the harmful effect of index misassignment that occurs with combinatorial approaches.
Solution Approach 2:
The patent uses universal adapter sequences as intermediaries between the index sequences and the nucleic acid fragments. These universal adapters mediate the connection, allowing standardized indexing while preventing direct cross-contamination between samples. The universal adapters serve as a buffer that maintains sample integrity during the pooling and sequencing process.
Data Source
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.