Transposase-Mediated Nucleic Acid Insertion for Sequencing
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Solution Overview
Problem
Current DNA sequencing technologies face challenges with short read lengths and issues like allelic dropout, template switching, and chimera formation, particularly in genome assembly, due to cumbersome dilution-based molecule labeling methods.
Innovation Solution
The use of transposase-mediated insertion of nucleic acid insert sequences into multiple sites within individual molecules to generate long contiguous DNA molecules, facilitating accurate and uniform amplification of genomic samples, including those from a single cell, through RNA transcription and subsequent reverse transcription, which reduces chimeric artifacts and sequencing errors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If dilution-based molecule labeling methods are used to address short read lengths, then genome assembly capability is improved, but allelic dropout, template switching and chimera formation occur
Solution Approach 1:
The genome is divided into multiple fragments that are individually tagged with transposon sequences. Each fragment is sequenced separately and then computationally assembled using the transposon insertion sites as anchors, enabling long-range genome assembly without physical long reads
Solution Approach 2:
Transposon sequences serve as intermediary markers inserted at random positions throughout the genome. These intermediaries provide unique identifiers that link short read sequences to their original genomic locations, enabling accurate assembly without requiring long read lengths
2Quantity of substance
If PCR or phi-29 based amplification methods are used to amplify genomic samples, then sufficient material for sequencing is obtained, but errors are amplified and propagated throughout the process
Solution Approach 1:
Transposon sequences are inserted into the genome before amplification. These pre-inserted markers serve as templates for subsequent amplification steps, ensuring that the amplification process starts from marked positions and maintains fidelity to the original genomic structure
Solution Approach 2:
The method uses multiple copies of transposon sequences inserted at different genomic positions as templates for amplification. Each transposon-flanked region is independently amplified and sequenced, allowing error correction through consensus sequencing of multiple copies
3Productivity
If standard genome sequencing methods are used, then sequencing throughput is achieved, but short read lengths pose a challenge to genome assembly
Solution Approach 1:
The method changes the parameter of read length indirectly by using computational assembly of short reads anchored by transposon insertion sites. This allows standard short-read sequencers to achieve long-range assembly capability through the use of transposon markers as positional references
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 method enables highly accurate and uniform amplification of nucleic acid samples, effectively addressing the limitations of existing methods by reducing chimeric artifacts and sequencing errors, and allowing for precise mapping of repetitive regions, even from small sample sizes, with minimal bias and high coverage.
Implementation Method 1
transposase mediated (and other types of enzyme-mediated) insertion of a nucleic acid insert sequence into a plurality of sites within individual molecules of a nucleic acid sample
Implementation Method 2
The sequence is used to direct RNA transcription of adjacent nucleic acid sequence into RNA
Implementation Method 3
reverse-transcribed into DNA that can be amplified or sequenced by downstream methods
Data Source
AI summary
Methods, compositions and kits are provided herein for insertional modification of nucleic acids by, for example transposase-mediated covalent insertion of insertion sequence into a sample nucleic acid molecule. Using sequence of the insertion to direct amplification of adjacent nucleic acid sequence, and using bar codes to map amplified sequence to partitions, one can map sample nucleic acid sequence to single molecules of the nucleic acid sample that are derived directly from the sample nucleic acid molecule.