Hierarchical Index Sequences for Accurate Multiplex Sample Assignment
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Solution Overview
Problem
Existing multiplex sequencing methods face challenges in accurately assigning fragment sequences to samples due to barcode errors, leading to cross-contamination and inefficient use of nucleotide length, which is exacerbated by the need for increased inter-barcode distance.
Innovation Solution
A computer-implemented method generates a set of oligonucleotides with nested subsets of index sequences, where higher tier subsets have greater sequence distances than lower tier subsets, allowing for error correction and optimized assignment quality while minimizing nucleotide sequencing costs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the barcode length is increased to maximize inter-barcode distance, then cross-contamination is reduced, but the length of the sequenced fragment is reduced
Solution Approach 1:
The index sequence is divided into multiple segments or tiers (first tier, second tier, third tier) with different sequence distances. Higher tiers have greater sequence distances for error correction, while lower tiers have shorter lengths for cost efficiency. This segmentation allows the system to achieve both high reliability and long fragment sequencing.
Solution Approach 2:
Different portions of the index sequence have different properties: the first tier indices have high sequence distance for error correction, while subsequent tiers have lower sequence distance but shorter length. This local differentiation optimizes both error correction capability and sequencing cost.
2Measurement precision
If the inter-barcode distance is increased to correct errors, then assignment accuracy is improved, but the number of nucleotides required increases
Solution Approach 1:
The index sequence is segmented into tiers with different sequence distances. Only the first tier requires high sequence distance for error correction, while subsequent tiers use shorter sequences. This reduces the total nucleotide count while maintaining assignment accuracy through the hierarchical error correction structure.
Solution Approach 2:
Instead of requiring all indices to have maximum sequence distance, the patent applies partial error correction by using only the first tier for primary error correction and allowing subsequent tiers to have lower sequence distances. This reduces the total nucleotide requirement while maintaining sufficient accuracy.
3Reliability
If a single high-distance barcode set is used for all samples, then error correction is maximized, but the efficiency and cost for experiments with fewer samples decreases
Solution Approach 1:
The patent enables dynamic selection of index sequences from different tiers based on the specific experimental needs. For experiments with fewer samples, only the first tier with high sequence distance is used, optimizing for error correction. For larger experiments, additional tiers can be incorporated. This dynamic adaptability maximizes both reliability and efficiency.
Solution Approach 2:
The hierarchical index structure serves multiple functions: the first tier provides error correction for all experiments, while subsequent tiers provide additional sample identification capacity for larger experiments. This multi-functionality allows a single system to optimize for both small and large-scale experiments.
Data Source
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AI summary
The present invention relates to a set of oligonucleotides comprising index sequences and wherein the set comprises a plurality of subsets of oligonucleotides with different index sequences, wherein the index sequences of a subset of oligonucleotides differ at least by a non-zero number of sequence changes from each other; and wherein the set comprises at least 2 hierarchical tiers of subsets, wherein index sequences of a higher tier subset are members of a lower tier subset, and wherein the index sequences of a lower tier subset differ by a lower minimum number of sequence changes from each other than the index sequences of a higher tier subset; and wherein the oligonucleotides are assigned to one or more subsets. The invention further relates to methods of generating and using such sets.