Long-Read Nucleic Acid Libraries with Transposome Tagging
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Solution Overview
Problem
Current nucleic acid library preparation methods for next-generation sequencing are inefficient, tedious, and costly, often requiring multiple steps and expensive instruments, and can result in underrepresentation of certain genomic portions.
Innovation Solution
A method involving immobilized transposomes on a solid support, such as beads, to fragment and tag nucleic acids, followed by amplification and addition of library adapters, with optional mutagenesis and enrichment steps using selection probes targeting specific sequences like MHC genes, to generate long read nucleic acid libraries.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If transposome-based methods are used to fragment and tag nucleic acids, then library preparation time and cost are reduced, but certain portions of the genome may be underrepresented in the libraries
Solution Approach 1:
The patent applies local quality by using different transposome densities for different genomic regions. Low-density transposomes are used for regions requiring long reads (e.g., MHC locus) to maintain representation accuracy, while high-density transposomes are used for other regions to maximize preparation efficiency. This spatial variation in transposome density resolves the contradiction between speed and accuracy.
Solution Approach 2:
The patent segments the library preparation process into distinct pathways: a long-read pathway using low-density transposomes for difficult-to-sequence regions, and a standard pathway using high-density transposomes for other regions. This segmentation allows each pathway to be optimized for its specific purpose, resolving the contradiction between overall efficiency and regional accuracy.
2Manufacturing precision
If multiple steps and expensive instruments are used for nucleic acid library preparation, then sequencing accuracy is improved, but preparation time and cost increase
Solution Approach 1:
The patent merges fragmentation and adapter tagging into a single transposome-based step, eliminating the need for separate fragmentation and end-repair steps. This consolidation maintains sequencing accuracy while dramatically reducing preparation time and eliminating the need for expensive fragmentation instruments.
Solution Approach 2:
The transposome system performs multiple functions simultaneously: it fragments the DNA, adds adapters, and introduces barcodes in a single self-contained reaction. This self-service approach eliminates the need for multiple separate instruments and steps, reducing both time and complexity while maintaining accuracy.
3Productivity
If high density of transposomes is used on beads, then fragmentation efficiency is improved, but average length of polynucleotides decreases
Solution Approach 1:
The patent makes the transposome density dynamic by allowing users to select between low-density and high-density bead preparations depending on the application. For long-read sequencing, low-density beads are used to preserve polynucleotide length, while for standard applications, high-density beads provide efficient fragmentation. This dynamic adjustment resolves the contradiction between efficiency and length.
Solution Approach 2:
The patent changes the key parameter of transposome density to control the outcome of the reaction. By adjusting this single parameter, the system can produce either long polynucleotides (low density) or efficiently fragmented DNA (high density), allowing optimization for different sequencing goals without changing the fundamental methodology.
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 reduces preparation time and cost, enhances representation of underrepresented genomic regions, and allows for efficient generation of long read nucleic acid libraries with improved sequencing coverage and accuracy.
Implementation Method 1
A method involving immobilized transposomes on a solid support, such as beads, to fragment and tag nucleic acids
Implementation Method 2
amplifying the plurality of polynucleotides to obtain amplified polynucleotides
Data Source
AI summary
Some embodiments of the methods and compositions provided herein relate to obtaining long read information from short reads of a target nucleic acid. Some embodiments include steps to selectively generate, mark, and amplify long nucleic acid fragments. Some embodiments include enriching for certain sequences in the long fragments with selection probes directed to major histocompatibility complex (MHC) genes. Some embodiments also include fragmenting the long nucleic acid fragments into shorter fragments for sequencing, and informatically reconstructing a sequence of the target nucleic acid.