Linker-Free Segmented Barcodes for Haplotype Phasing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current linked-read sequencing technologies face challenges such as high costs, complex instrumentation, barcode collisions, and incompatibility with standard sequencing protocols, limiting their adoption for haplotype information retrieval and multiplexing capabilities.
Innovation Solution
A novel method involving solid supports with linker-free segmented barcodes, where each solid support has multiple copies of single-stranded DNA oligonucleotides with attached barcode segments, allowing for efficient barcode diversity and error detection, compatible with standard Illumina sequencing protocols.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If standard short-read sequencing is used, then high throughput and low cost are achieved, but contiguity information and haplotype data are lost
Solution Approach 1:
The DNA molecule is segmented into multiple fragments, each tagged with a barcode during tagmentation. These barcoded fragments are then sequenced separately using standard short-read technology. The segmentation allows each fragment to be processed independently while the barcode preserves the original molecular context, resolving the contradiction between high-throughput short-read sequencing and contiguity information preservation.
Solution Approach 2:
A barcode sequence acts as an intermediary between the DNA fragment and the sequencing platform. The barcode is attached to each fragment during tagmentation and serves as a molecular identifier that links short reads back to their parent long DNA molecule. This intermediary enables standard short-read sequencers to generate data that can be computationally assembled into long-range haplotype information.
2Loss of information
If existing linked-read technologies are used, then long-range haplotype information is preserved, but high costs and complex instrumentation are required
Solution Approach 1:
The tagmentation-based barcoding system is designed to be universal and compatible with standard Illumina sequencing platforms. The same tagmentation reaction and sequencing workflow can process both standard libraries and haplotype-preserving libraries without requiring specialized instruments. This multi-functionality allows the system to preserve haplotype information while using conventional, cost-effective sequencing infrastructure.
Solution Approach 2:
The invention uses disposable barcoded adapters and standard consumables rather than expensive, reusable specialized instruments. The barcoding is achieved through inexpensive tagmentation reactions using commercially available Tn5 transposase and custom barcoded adapters. This approach replaces costly dedicated long-read or linked-read instrumentation with affordable, disposable molecular tags that work on standard sequencers.
3Loss of information
If existing linked-read technologies are used, then haplotype information is preserved, but barcode collisions and incompatibility with standard protocols occur
Solution Approach 1:
The barcode design incorporates multiple dimensions of diversity: unique molecular identifiers (UMIs) on each adapter, combinatorial indexing with multiple index sequences, and positional information from the tagmentation pattern. This multi-dimensional barcoding strategy dramatically increases the effective barcode space, reducing collision probability while maintaining compatibility with standard dual-indexed Illumina sequencing protocols.
Solution Approach 2:
The system optimizes barcode parameters including length, diversity, and error correction capabilities. By carefully designing barcode sequences with sufficient length and incorporating error correction codes, the system achieves extremely low collision rates. The barcode parameters are tuned to work within the constraints of standard Illumina sequencing while maximizing uniqueness and reliability.
4Reliability
If comprehensive barcode diversity is implemented, then barcode collisions are reduced, but manufacturing complexity increases
Solution Approach 1:
Comprehensive barcode diversity is pre-established during the solid support manufacturing phase through combinatorial chemistry or oligo synthesis. Each bead is synthesized with a unique barcode combination from a large predefined library. This preliminary action ensures that when samples are processed, the full barcode diversity is already in place, eliminating the need for complex runtime barcode generation or management systems.
Solution Approach 2:
The comprehensive barcode is segmented into multiple components (UMI, sample index, bead index) that are synthesized and assembled separately before final bead production. This segmentation allows each component to be optimized independently and simplifies the manufacturing process by breaking down the complex barcode synthesis into manageable steps that can be performed using standard oligo synthesis and bead coupling techniques.
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 enables high-throughput, cost-effective generation of sequencing libraries with reduced barcode collisions, preserving contiguity information and facilitating multiplexing, thus improving the accuracy and efficiency of haplotype analysis.
Implementation Method 1
bead-immobilized TN5 transposase
Data Source
AI summary
The present invention relates to methods for producing solid supports. The present invention further provides a mixture of said solid supports for tagmentation of target DNA for DNA sequencing approaches, a corresponding kit comprising the same and methods employing said mixture of solid supports and/or kit. Specifically, methods for producing sequencing libraries and corresponding DNA sequencing methods for analyzing the generated sequencing libraries and tools used therein are provided. In particular, DNA In sequencing approaches allowing preservation of contiguity information of long DNA fragments even when using short read sequencing approaches are disclosed. A key concept of the present invention is to employ segmented barcodes, with every barcode segmented allowing for barcode error detection and correction on a segment level. Preferred barcode sequences employed are characterized in that they comprise no linker sequences or only linker sequences of one or two nucleotides in length between the barcode segments.