Modular Nucleic Acid Adapters for NGS Multiplexing
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Solution Overview
Problem
Current nucleic acid adapters for next-generation sequencing (NGS) face challenges such as high manufacturing costs, inefficiencies in sequencing, and inaccuracies in sample identification and variant calling due to crossover effects and error-prone unique identifiers (UIDs).
Innovation Solution
The development of modular nucleic acid adapters with decoupled unique identifiers (UIDs) and sample identifiers (SIDs), where UIDs are distributed on separate oligos and SIDs are incorporated through PCR, reducing the number of necessary oligos and using variable length punctuation marks to enhance sequencing complexity without PhiX spike-in, and incorporating SIDs in both PCR primers for improved accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If forked adapters with UIDs and SIDs are used for sample multiplexing and molecular counting, then sequencing efficiency and accuracy are improved, but manufacturing cost increases due to the large number of different oligos required
Solution Approach 1:
The adapter is divided into modular components: a universal adapter sequence and variable UID/SID elements. This segmentation allows the universal portion to be manufactured once and reused, while only the variable portions need to be synthesized in multiple versions, significantly reducing the total number of oligos that must be produced.
Solution Approach 2:
The universal adapter sequence serves multiple functions: it provides the binding site for NGS platforms, enables PCR amplification, and supports various UID/SID combinations. This multi-functionality allows a single universal sequence to work with numerous sample identifiers, reducing the need to manufacture separate oligos for each combination.
2Quantity of substance
If 2-base UIDs are used, then the number of possible unique identifiers increases, but error differentiation capability decreases leading to over counting of molecules
Solution Approach 1:
The UID length parameter is optimized to 4 bases instead of 2 bases. This parameter change provides 256 possible combinations (4^4) compared to only 16 combinations (4^2), while maintaining sufficient error differentiation capability. The increased length provides a margin of safety against sequencing errors while still providing adequate multiplexing capacity.
3Device complexity
If SID is attached to only one side of the adapter and not directly to the molecule of interest, then adapter design is simplified, but crossover effects occur resulting in incorrect sample assignment and variant calling errors
Solution Approach 1:
The SID is merged with the universal adapter sequence on both the forward and reverse strands. This combining ensures that both strands of the DNA fragment carry the same sample identifier information, preventing crossover effects where strands from different samples might be incorrectly paired while maintaining a relatively simple overall adapter structure.
4Measurement precision
If PhiX is added to sequencing libraries to increase complexity at positions 3 and 4, then sequencing quality improves, but the number of sequencing reads available for sample molecules decreases
Solution Approach 1:
Instead of adding PhiX globally to all libraries, the punctuation mark sequence is locally integrated into the adapter design at positions 3 and 4. This local quality enhancement provides the necessary sequence complexity exactly where needed for accurate base calling, without the overhead of adding separate PhiX control molecules that would consume sequencing reads.
Data Source
AI summary
The present disclosure provides a kit for preparing a library of nucleic acids. The kit includes first and second oligonucleotide, each having a tail sequence, a common sequence, and at least one of a unique identifier sequence, and a variable length punctuation mark. The kit further includes a first primer having a first sample identifier sequence and a first priming sequence at a 3′ end of the first primer. The first priming sequence includes the tail sequence of the first oligonucleotide. The kit further includes a second primer having a second sample identifier sequence and a second priming sequence at a 3′ end of the second primer. The second priming sequence is complimentary to the second tail sequence of the second oligonucleotide.