Partitioned DNA Library Prep via Segmentation
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Solution Overview
Problem
Current library preparation methods for next-generation sequencing face challenges such as amplification bias, artifact production, and high computational burden, leading to inaccurate results and false positives, particularly in applications like immune repertoire sequencing and microbiome analysis.
Innovation Solution
The use of functionalized particles distributed across partitions for library preparation, which reduces amplification bias and artifact production by limiting competition during amplification reactions, allowing for accurate library generation without extensive computational corrections and specialized equipment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If PCR amplification is used to amplify trace amounts of DNA, then sequencing library generation is enabled, but amplification bias occurs leading to unequal sequencing read distributions
Solution Approach 1:
The patent divides the amplification process into separate stages: initial amplification of individual templates to create molecular bars, followed by pooling and secondary amplification. This segmentation prevents competition bias by ensuring each template is amplified independently first, then combined, eliminating the unequal read distribution problem of conventional single-stage PCR.
Solution Approach 2:
The patent introduces molecular bars as intermediary elements that tag each original template molecule. These bars serve as identifiers that allow tracking and equalization of reads back to original templates, mediating between the amplification process and the sequencing analysis to correct for bias.
2Quantity of substance
If PCR amplification is performed to generate sequencing libraries, then trace DNA amounts are amplified, but artifacts such as chimeric and heteroduplex molecules are produced
Solution Approach 1:
By segmenting amplification into two distinct stages with molecular bar tagging, the patent prevents chimera formation. Each template is amplified separately with its unique bar, then pools are created. This eliminates the mixing and recombination artifacts that occur in conventional single-stage PCR where templates compete and recombine.
Solution Approach 2:
The molecular bars provide feedback information about the original template identity and abundance. This feedback allows computational correction of remaining artifacts and ensures accurate representation of original samples, enabling detection of very low allelic fractions that would be obscured by artifacts in conventional methods.
3Productivity
If conventional PCR workflows are used for immune repertoire sequencing, then library preparation is completed, but accuracy falls below 70% and receptor frequencies are overestimated
Solution Approach 1:
The patent applies segmentation to immune repertoire sequencing by performing initial amplification with molecular bars on individual VDJ templates, then pooling and performing secondary amplification. This two-stage process maintains accuracy above 90% by preventing the competition and recombination artifacts that cause conventional single-stage PCR to overestimate receptor frequencies by up to 5000-fold.
Solution Approach 2:
Molecular bars serve as intermediaries that track original template identities through the amplification process. This allows accurate counting and frequency calculation of immune receptors by referencing back to the original templates, eliminating the estimation errors inherent in conventional methods.
4Reliability
If UMIs are used for post-sequencing correction, then amplification errors are reduced, but false UMIs are introduced and deeper sequencing coverage is required
Solution Approach 1:
Instead of introducing UMIs after sequencing and performing complex computational correction, the patent performs preliminary action by adding molecular bars during the amplification process itself. This preliminary tagging eliminates errors at the source, reducing false positives before sequencing occurs, thereby avoiding the need for deep re-sequencing and complex computational filtering.
Solution Approach 2:
The patent skips the complex post-sequencing computational correction step by preventing errors during amplification through molecular bar tagging. This rushing through of the correction process eliminates the need for deep re-sequencing and extensive computational filtering, reducing both time and computational resources required.
Data Source
AI summary
The disclosure provides compositions, methods, and systems for extremely accurate generation of nucleic acid libraries, without use of bulk amplification methods. Accurate library preparation is achieved in a rapid manner, with respect to sample partitioning and amplification in a manner that achieves high performance in relation to low levels of amplification bias and low levels of artifact/chimeric sequence generation. Implementation of methods described also achieve library preparation with significantly reduced false positive rates, across a wide variety of applications.