Asymmetric DNA Library Generation for Duplex Sequencing
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Solution Overview
Problem
Conventional DNA sequencing methods struggle to accurately detect low incidence mutations due to high background error rates in sequencing and DNA amplification, particularly in single-cell sequencing, where errors introduced during first-strand synthesis are propagated and difficult to distinguish from genetic variants.
Innovation Solution
A method for generating a DNA library with asymmetric molecular identifier sequences on both ends of DNA fragments using adaptors with different nucleotide sequences, allowing for the alignment and comparison of both strands to identify and correct errors, without requiring Y-adaptors or costly modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If single-cell sequencing or single-stranded molecular barcoding is used to detect low incidence mutations, then the ability to sequence DNA from single cells is improved, but the reliability of mutation detection deteriorates due to high background error rates from DNA polymerase misinsertions that cannot be distinguished from true mutations
Solution Approach 1:
The DNA molecule is segmented into two separate strands, each independently tagged with molecular identifiers. By sequencing and comparing both strands separately, the method divides the error detection problem into two independent verification paths, allowing errors in one strand to be identified and corrected using the other strand as reference.
Solution Approach 2:
The original DNA strand is copied to create a complementary strand, and both the original and copy are independently tagged and sequenced. This creates redundant information where true mutations appear in both copies while polymerase errors appear in only one, enabling error identification and correction through comparison.
2Reliability
If Y-adaptors or forked adaptors are used to introduce asymmetry for duplex sequencing, then the ability to track both strands is improved, but the device complexity and manufacturing difficulty increase due to required structural modifications and additional synthesis steps
Solution Approach 1:
Instead of using structurally complex Y-adaptors, the method introduces asymmetry through sequential tagging: the first adaptor is ligated to one end of the DNA fragment, and the second adaptor is ligated to the other end. This creates asymmetric molecular identifiers on each strand without requiring complex adaptor structures, simplifying the overall system while maintaining the ability to track both strands.
Solution Approach 2:
The first adaptor is ligated and its sequence is incorporated into the DNA fragment before the second adaptor is added. This preliminary action ensures that each end of the DNA fragment has a distinct molecular identifier from the outset, eliminating the need for complex simultaneous tagging mechanisms required by Y-adaptors.
3Ease of operation
If conventional adaptors are used without asymmetric tagging, then the simplicity of the sequencing process is maintained, but the ability to detect and correct sequencing errors deteriorates
Solution Approach 1:
Molecular identifiers act as intermediary tags that link each sequenced read back to its original DNA strand. These intermediaries enable the comparison and consensus-building process without complicating the core sequencing operation, as the identifiers are simply appended sequences that facilitate error detection through bioinformatic analysis.
Data Source
AI summary
Methods and products are disclosed for asymmetrically adapting fragmented nucleic acids for next generation sequencing, including providing strand identifier sequences and index sequences to identify the source strand and sample, respectively, of the fragmented nucleic acids. The methods and products allow for efficient and reliable detection of low-frequency mutations including in subpopulations of cells within a subject and also for the amplification of the fragmented nucleic acids when there is a low yield of isolated fragmented nucleic acids.