High Coverage stLFR Sequencing via Staggered Breaks
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Solution Overview
Problem
Current methods for determining the sequence of target nucleic acids lack information on the order of single to multi-base variants transmitted as contiguous blocks on homologous chromosomes.
Innovation Solution
A method involving the preparation of a library of barcoded polynucleotides, where fragments derived from the target nucleic acid are processed to introduce staggered single-stranded breaks, and capture oligonucleotide sequences with barcodes are associated with the subfragments, allowing for amplification and further processing to generate a sequencing library.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If co-barcoding technology is used to determine sequence information, then sequencing coverage is improved, but information on the order of variants transmitted as contiguous blocks is lost
Solution Approach 1:
The patent divides the long DNA molecule into multiple sub-fragments through controlled shearing or enzymatic digestion, while preserving the relative order information through spatial positioning on the bead surface. Each sub-fragment is then individually barcoded and sequenced, allowing reconstruction of the original sequence order through computational assembly of the spatially-resolved barcode data.
Solution Approach 2:
The patent introduces spatial dimensionality by attaching multiple barcodes to different locations on a single bead surface, where the spatial position of each barcode relative to others encodes the order information. This transforms the problem from a one-dimensional linear sequence problem into a two-dimensional spatial arrangement problem that can be resolved through imaging and coordinate mapping.
2Measurement precision
If long DNA molecules are fragmented for sequencing, then sequencing accuracy is improved, but the contiguous block structure of variants is disrupted
Solution Approach 1:
The patent uses barcodes as intermediary markers that are attached to each sub-fragment. These barcodes serve as molecular coordinates that record the original position and order of fragments within the contiguous block. Even though the physical continuity is broken by fragmentation, the barcode intermediaries preserve the structural information needed to reconstruct the original arrangement.
Solution Approach 2:
The patent creates multiple copies of barcode information on each bead, with each copy associated with a specific sub-fragment. These barcode copies act as informational replicas that preserve the original sequence order data, allowing the contiguous block structure to be computationally reconstructed from the fragmented pieces through barcode matching and spatial coordinate analysis.
Data Source
AI summary
Described herein are high coverage single tube Long Fragment Read (stLFR) technology which uses performs stLFR on target DNA fragments that have already been amplified before they are co-barcoded, which provides higher amount of DNA for sequencing and increases sequencing coverage. In some embodiments, the high coverage stLFR described in this application uses two rounds of stLFR. In some embodiments, the target DNA fragments are transposed with transposons having particular positional barcodes that can be used to order sequence reads.