Sequencing Primer Design for Adapter Dimer Detection in Libraries
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Solution Overview
Problem
Adapter dimers formed during nucleic acid library preparation contaminate sequencing results, leading to lower quality data due to their faster amplification and mismatch issues, which existing quantification techniques fail to address effectively.
Innovation Solution
A sequencing workflow that uses design-guided sequencing primers to generate adapter dimer sequencing data, allowing for quality metrics calculation and identification of libraries with excessive adapter dimers, enabling rebalancing and normalization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If adapter dimers are not removed during library preparation, then sequencing throughput is maintained, but sequencing quality deteriorates due to adapter dimer contamination
Solution Approach 1:
The patent performs adapter dimer detection and identification before the main sequencing run by sequencing a portion of the library to generate initial data, identifying adapter dimers based on their characteristic sequences and lengths, and determining their abundance. This preliminary characterization allows for optimized library preparation and rebalancing before full sequencing, preventing adapter dimer contamination from compromising sequencing quality while maintaining throughput.
2Reliability
If adapter dimers are detected and removed, then sequencing quality improves, but library preparation time increases
Solution Approach 1:
The patent employs a universal adapter sequence that is used across different library preparations and sequencing runs. This universal adapter allows for consistent adapter dimer detection and characterization across multiple libraries and experiments. The same sequencing primer and detection methodology can be applied universally to identify and quantify adapter dimers in any library prepared with this adapter design, streamlining the process and reducing the time needed for quality assessment.
Solution Approach 2:
The patent uses sequencing to generate copies of adapter dimer sequences for analysis. By sequencing adapter dimers and generating their sequence data, the method creates digital copies that can be analyzed in silico to identify adapter dimer characteristics, abundance, and impact on library quality. This computational analysis of sequence copies allows for rapid assessment without requiring physical removal or manipulation of the actual adapter dimers.
3Quantity of substance
If adapter dimers are amplified during library amplification, then library yield increases, but adapter dimer contamination increases
Solution Approach 1:
The patent implements a feedback mechanism where adapter dimers are detected and quantified in the library preparation process. The abundance of adapter dimers is determined by sequencing and analyzing the generated data. This information feeds back into the library preparation process, allowing for adjustment of amplification conditions, adapter concentrations, or library normalization to reduce adapter dimer formation while maintaining acceptable library yield. The feedback loop enables optimization of the balance between yield and contamination.
Solution Approach 2:
The patent utilizes changes in sequencing parameters and analysis parameters to detect and characterize adapter dimers. By adjusting sequencing depth, read length, or analysis thresholds, the method can sensitively detect adapter dimers even at low abundances. The patent also employs parameter changes in library preparation, such as modifying adapter concentrations, ligation conditions, or amplification cycles, based on the detected adapter dimer levels to optimize the balance between library yield and contamination.
Data Source
AI summary
A library sequencing technique with library quality control metrics is described. Sequence data using a sequencing primer that is complementary to a common adapter sequence in fragments of a nucleic acid sequencing library. The sequencing primer excludes a 3′ terminal nucleotide of the common adapter sequence at a junction with a fragment insert. This exclusion avoids a mismatch region in any adapter dimers present in the sequencing library, and the sequence data includes adapter dimer sequence data, which is used to generate the quality control metrics.