Next-Generation Sequencer Primer Index Design for Read Consistency
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Solution Overview
Problem
Next-generation sequencers using primers with indices face significant variations in the number of reads, leading to inaccurate and inefficient analysis due to the properties of the index sequence, which current techniques fail to systematically address.
Innovation Solution
A primer design for next-generation sequencers is developed, featuring a nucleotide sequence with an index sequence that utilizes an estimation formula to predict and achieve a putative number of reads exceeding 50,000, specifically using the sequence 5'-CAAGCAGAAGACGGCATACGAGAT(N 5 to 15)-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG, where N represents an index sequence of 5 to 15 nucleotides, and the formula calculates the number of reads based on nucleotide types and positions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If an index sequence is added to the primer for next-generation sequencing, then multiple samples can be distinguished and analyzed simultaneously, but the number of reads varies significantly depending on the index sequence properties
Solution Approach 1:
The patent applies parameter changes by systematically varying the nucleotide composition and position of index sequences to identify optimal parameters. The estimation formula calculates the putative number of reads based on nucleotide types (A, C, G, T) at each position (1-15) of the index sequence, allowing selection of index sequences with parameters that ensure sufficient read numbers (≥50,000 reads) while maintaining multiplexing capability.
2Productivity
If conventional primers with arbitrary index sequences are used, then various samples can be multiplexed, but the accuracy of DNA analysis decreases due to significant variation in read numbers
Solution Approach 1:
The patent applies preliminary action by calculating the putative number of reads for each candidate index sequence using the estimation formula before actual sequencing. This allows selection of index sequences with predicted sufficient read numbers (≥50,000) in advance, ensuring both high throughput and accurate analysis without needing to resequence samples with insufficient reads.
3Ease of manufacture
If no systematic analysis of index sequence properties is performed, then primer design is simple, but the accuracy and reliability of next-generation sequencing analysis is insufficient
Solution Approach 1:
The patent applies self-service by providing an automated estimation formula that calculates the putative number of reads based on index sequence properties. This formula serves as a self-evaluation tool that guides primer design, allowing researchers to systematically select optimal index sequences without complex experimental optimization, thereby improving reliability while maintaining ease of use.
Data Source
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AI summary
This invention provides a primer used for a next-generation sequencer that can provide a large number of reads. On the basis of the sequence: 5'-CAAGCAGAAGACGGCATACGAGAT-N5 to 15-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3', wherein N5 to 15 indicates an index sequence of 5 to 15 nucleotides), an index sequence is designed as a nucleotide sequence exhibiting the putative number of reads, which is calculated using the estimation formula designating the number of reads as a purpose variable and the type of nucleotides in the index sequence as an explanatory variable, exceeding a given level.