Barcoded DNA Transposition for Contiguity-Preserving Sequencing
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Solution Overview
Problem
Existing nucleic acid sequencing methods face challenges in maintaining contiguity and efficiently determining phasing and methylation status of target nucleic acid sequences, particularly in complex samples like formalin fixed paraffin embedded (FFPE) samples and cell-free DNA.
Innovation Solution
The use of transposome complexes with transferred and non-transferred strands, combined with solid supports having immobilized oligonucleotides with unique barcode sequences, allows for the fragmentation and tagging of nucleic acids while preserving contiguity, enabling simultaneous determination of phasing and methylation status without additional purification steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If nucleic acid is fragmented for sequencing, then sequencing efficiency is improved, but contiguity information is lost
Solution Approach 1:
The nucleic acid is divided into multiple fragments through transposition, with each fragment receiving a barcode tag. This segmentation enables parallel processing of multiple fragments while the barcode preserves the original contiguity relationship, allowing reconstruction of the full sequence from fragmented data.
Solution Approach 2:
A barcode sequence acts as an intermediary between the fragmented nucleic acid pieces and the contiguity information. The barcode is attached to each fragment and serves as a identifier that links fragments back to their original position in the parent molecule, enabling reconstruction without physical continuity.
2Measurement precision
If multiple purification steps are performed to remove transposases and other components, then sequencing quality is improved, but processing time and complexity increase
Solution Approach 1:
The transposase enzyme is extracted and removed from the reaction mixture after the transposition reaction is complete. This extraction eliminates the need for complex purification steps to remove active enzymes that could interfere with downstream sequencing operations, reducing both time and complexity while maintaining sequencing quality.
3Loss of information
If transposome complexes are used for fragmentation and tagging, then contiguity preservation is improved, but device complexity increases
Solution Approach 1:
Multiple functions are merged into the transposome complex: fragmentation of nucleic acid, attachment of barcode tags, and preservation of contiguity information all occur in a single integrated reaction step. This combining of functions reduces the need for multiple separate operations and simplifies the overall workflow despite the complexity of the individual transposome structure.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach maintains contiguity and provides efficient sequencing data, particularly in challenging samples, enhancing the detection of genomic variants and methylation patterns without the need for additional purification steps.
Implementation Method 1
at least one of the transposons of the transposome complex comprises an adaptor sequence capable of hybridizing to a complementary capture sequence
Data Source
AI summary
Embodiments provided herein relate to methods and compositions for preparing an immobilized library of barcoded DNA fragments of a target nucleic acid, identifying genomic variants, determining the contiguity information, phasing information, and methylation status of the target nucleic acid.