Positional Co-Barcoding of Long DNA for Short-Read Sequencing
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Solution Overview
Problem
Sequencing long genomic DNA is challenging due to inefficiencies in existing platforms, such as DNBseq and Illumina sequencers, which limit read lengths and struggle with highly repetitive sequences, making it difficult to accurately decipher sequence reads from long DNA molecules.
Innovation Solution
Methods for producing single-stranded and double-stranded adaptered constructs without nanodrops, involving nested sets of nucleic acid constructs with unique barcodes and varying lengths, and processes like circularization and controlled extension to generate adaptered fragments for sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If bridge amplification is used on Illumina sequencers to sequence long DNA molecules, then sequencing can be performed on these molecules, but the amplification efficiency is poor and read length is limited to less than 500 bases
Solution Approach 1:
The patent segments long DNA molecules into multiple shorter fragments through controlled nick translation, creating a nested set of fragments with different lengths. Each fragment is then independently barcoded and sequenced, allowing the reconstruction of the original long molecule sequence from multiple short reads. This segmentation resolves the contradiction by making long molecules compatible with short-read sequencing technology.
Solution Approach 2:
The patent creates a nested set of DNA fragments where shorter fragments are contained within the context of longer fragments. Multiple nested sets with different barcode sequences allow hierarchical organization of sequence data, enabling accurate reconstruction of long DNA sequences from nested collections of short reads while maintaining positional information.
2Productivity
If standard sequencing methods are used for highly repetitive sequences, then sequencing can be performed, but it is difficult to decipher whether reads are from different positions or duplicates of the same position
Solution Approach 1:
The patent applies preliminary barcoding to DNA fragments before sequencing, assigning unique positional identifiers to each fragment. This preliminary action of tagging fragments with barcodes that encode their original positions allows accurate reconstruction of repetitive sequences by matching barcodes to their source locations, resolving the ambiguity of whether reads come from different positions or are duplicates.
3Productivity
If DNBseq sequencers are used for long DNA molecules, then sequencing can be performed, but it is difficult to produce enough DNB copies for high-quality sequencing
Solution Approach 1:
The patent segments long DNA molecules into shorter fragments that are more amenable to DNB formation. By creating a nested set of shorter fragments from each long molecule, the system increases the number of sequencable units while maintaining the ability to reconstruct the original long sequence, thus improving DNB production efficiency without sacrificing long-read capability.
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
Enables accurate and efficient sequencing of long DNA molecules by generating nested sets of adaptered fragments with unique barcodes, allowing for complete coverage and positional information, even in repetitive sequences.
Implementation Method 1
the first and the second hybridization sequences are complementary to each other
Data Source
AI summary
The methods and compositions disclosed herein relate to preparing libraries to sequence long molecules in their entirety using massively parallel short read sequencing. The methods disclosed herein generate a nested set of nucleic acid constructs for each genomic fragment and generate a plurality of nested sets for a plurality of genomic fragments. The nucleic acid constructs may be single-stranded or double-stranded. Each nucleic acid construct in each nested set comprises a barcode and target sequence portion, and nucleic acid constructs within each nested set have different lengths. The nucleic acid constructs in each nested set share a unique barcode sequence.