Nucleic Acid Template Library Indexing for Accurate Haplotype Sequencing
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Solution Overview
Problem
Existing nucleic acid sequencing technologies face challenges in efficiently obtaining sequence information, particularly haplotype information, with high error rates and difficulties in distinguishing between different alleles, especially at high nucleic acid concentrations.
Innovation Solution
The use of transposomes with non-contiguous transposon sequences and transposases to compartmentalize and index nucleic acids, combined with methods to maintain physical proximity of fragmented nucleic acid fragments, allows for the preparation of template libraries that enable high-concentration sequencing with reduced error rates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If nucleic acid concentration is increased to improve sequencing productivity, then sequencing throughput increases, but error rate increases and haplotype distinction becomes difficult
Solution Approach 1:
The nucleic acid sample is divided into multiple separate reaction vessels, each containing a subset of the total nucleic acid molecules. This segmentation allows high-concentration sequencing without the errors and haplotype confusion that occur when all molecules are processed together in a single reaction.
Solution Approach 2:
Transposomes with non-contiguous transposon sequences serve as intermediary markers that are inserted into nucleic acid fragments. These transposomes maintain physical proximity information and enable accurate haplotype distinction even when nucleic acids are processed at high concentrations across multiple vessels.
2Measurement precision
If transposomes with non-contiguous transposon sequences are used to maintain physical proximity information, then haplotype sequencing accuracy improves, but device complexity increases
Solution Approach 1:
The transposome is segmented into non-contiguous transposon sequences that are inserted at different locations in the nucleic acid fragment. This segmentation allows the transposome to maintain physical proximity information across fragmented nucleic acids while enabling standard sequencing procedures.
Solution Approach 2:
The transposome structure serves multiple functions: it fragments the nucleic acid, inserts identifiable markers (transposon sequences), maintains physical proximity information, and enables subsequent sequencing. This multi-functionality reduces the need for separate devices or procedures.
3Reliability
If transposase is removed from the template nucleic acid after transposition, then sequencing error rate decreases, but additional processing steps are required
Solution Approach 1:
The transposase enzyme is removed from the template nucleic acid after the transposition reaction is complete. This extraction eliminates the source of sequencing errors caused by transposase binding, while the transposon sequences remain inserted in the nucleic acid to provide sequencing markers and physical proximity information.
Data Source
AI summary
Embodiments of the present invention relate to sequencing nucleic acids. In particular, embodiments of the methods and compositions provided herein relate to preparing nucleic acid templates and obtaining sequence data therefrom.