Single-Tube DNA Co-Barcoding With Bead Capture for Haplotype Sequencing
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Solution Overview
Problem
Existing genome sequencing technologies for determining the order of single to multi-base variants on homologous chromosomes are technically challenging, expensive, and lack unique co-barcoding, often requiring separate whole genome sequencing, limiting their use due to cost and ease of use.
Innovation Solution
The implementation of single tube Long Fragment Read (stLFR) technology, which uses microbeads with unique barcodes to co-barcoding sub-fragments of long DNA molecules, enabling efficient and cost-effective sequencing on standard devices like the BGISEQ-500, with a ligation-based combinatorial barcode generation strategy producing over 1.8 billion different barcodes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If co-barcoding technologies are implemented to determine variant order on homologous chromosomes, then haplotype information and structural variation detection are improved, but cost and technical complexity increase significantly
Solution Approach 1:
The patent segments the co-barcoding process into distinct functional components: (1) transposon-mediated fragmentation and tagging of long DNA molecules, (2) bead-based capture with unique barcodes, and (3) ligation-based barcode assignment. This segmentation allows each component to be optimized independently and simplifies the overall workflow compared to previous monolithic approaches.
Solution Approach 2:
The patent introduces beads as an intermediary carrier that bridges the gap between long DNA molecules and sequencing libraries. Each bead carries unique barcodes and captures specific DNA fragments through hybridization, serving as a mediator that enables co-barcoding without requiring complex direct labeling protocols.
2Measurement precision
If co-barcoding is performed to achieve accurate variant calling and haplotyping, then data quality is improved, but cost increases due to separate whole genome sequencing requirements
Solution Approach 1:
The patent merges the co-barcoding function with the whole genome sequencing library preparation into a single integrated workflow. The transposon-based fragmentation, bead capture, and barcode assignment all occur in one protocol, eliminating the need for separate co-barcoding and WGS experiments, thereby reducing costs while maintaining high variant calling accuracy.
Solution Approach 2:
The patent creates a universal library preparation protocol that simultaneously achieves multiple functions: (1) fragmentation of long DNA molecules, (2) addition of unique barcodes for co-barcoding, (3) preparation of sequencing-ready libraries, and (4) enrichment of haplotype information. This multi-functional approach replaces previous multi-step workflows requiring separate experiments.
3Loss of information
If traditional co-barcoding methods are used, then haplotype information can be obtained, but unique co-barcoding is lost and data quality decreases
Solution Approach 1:
The patent performs preliminary action by pre-attaching unique barcodes to beads before the DNA capture step. This ensures that each captured DNA fragment is immediately assigned a unique identifier, preventing barcode collisions and ensuring high-quality co-barcoding from the outset, rather than attempting to assign barcodes after capture.
Solution Approach 2:
The patent uses copying by attaching multiple copies of the same barcode sequence to each bead (approximately 400,000 copies per bead). This ensures robust signal detection during sequencing while maintaining unique identification of each original DNA molecule, thereby improving data quality without losing haplotype information.
4Measurement precision
If standard whole genome sequencing is performed separately, then variant calling is improved, but ease of use decreases and cost increases
Solution Approach 1:
The patent merges co-barcoding and whole genome sequencing into a single library preparation protocol. The transposon-based fragmentation and bead capture process simultaneously prepares samples for both haplotype-resolved sequencing and standard variant calling, eliminating the need for separate experiments and simplifying the workflow for users.
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
stLFR achieves high read coverage and accurate variant calling with reduced false positives and negatives, facilitating powerful informatics approaches for de novo assembly and structural variation detection, while maintaining a low cost per sample.
Implementation Method 1
The first step of stLFR involves inserting a hybridization sequence approximately every 200-1000 base pairs on long genomic DNA molecules. This is achieved using transposons.
Implementation Method 2
The transposon integrated DNA is then mixed with beads that each contain ~400,000 copies of an adapter sequence that contains a unique barcode shared by all adapters on the bead, a common PCR primer site, and a common capture sequence that is complementary to the sequence on the integrated transposons. After the genomic DNA is captured to the beads
Implementation Method 3
the transposons are ligated to the barcode adapters
Data Source
AI summary
Methods and compositions for preparing a nucleic acid sequencing library are described including (a) transposing an insertion sequence into first fragments of the target nucleic acid, wherein the insertion sequence comprises a hybridization sequence, and wherein the transposing produces nicks in the first fragments; (b) combining in a single mixture (i) the first fragments of the target nucleic acid from (a), (ii) a splint oligonucleotide, and (iii) a population of beads, wherein each bead comprises capture oligonucleotides immobilized thereon, and (c) ligating capture oligonucleotides of individual beads to inserted hybridization sequences of individual first fragments.