Long-Read Nucleic Acid Libraries with Bead-Bound Transposomes
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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 suppression PCR, and enrichment using selection probes to target challenging medically relevant genes.
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 resulting libraries
Solution Approach 1:
The patent applies local quality by using multiple transposomes with different sequences (e.g., Transposome A, Transposome B, Transposome C) that target different genomic regions with varying efficiencies. Each transposome type has specific sequence preferences, and by combining them, the method ensures more uniform coverage across diverse genomic portions while maintaining the efficiency benefits of transposome-based fragmentation and tagging.
2Measurement precision
If multiple steps and expensive instruments are used for nucleic acid library preparation, then sequencing accuracy is improved, but preparation complexity and cost increase
Solution Approach 1:
The patent merges multiple library preparation steps into a unified transposome-based workflow. The transposomes perform fragmentation, tagging, and adapter incorporation in a single integrated process, eliminating the need for separate instrumentation steps while maintaining sequencing accuracy through the inherent precision of the transposase enzyme mechanism and controlled reaction conditions.
Solution Approach 2:
The transposomes serve multiple functions simultaneously: they fragment the nucleic acid substrate, add adapter sequences for sequencing, and incorporate barcodes for sample multiplexing. This multi-functionality eliminates the need for separate instruments and steps that would otherwise be required for each function, reducing preparation complexity while maintaining accuracy.
3Productivity
If high density of transposomes is used on beads, then fragmentation efficiency is improved, but average fragment length decreases
Solution Approach 1:
The patent applies dynamics by controlling the density of transposomes on beads as a variable parameter. Different bead populations have different transposome densities (e.g., low density for longer fragments, high density for shorter fragments), and the method dynamically adjusts this density based on the desired fragment length distribution and application requirements, optimizing both fragmentation efficiency and fragment length control.
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 reduces preparation time and cost, enhances representation of underrepresented genomic regions, and allows the use of unpurified or degraded samples, improving sequencing efficiency and accuracy.
Implementation Method 1
nucleic acid fragment libraries may be prepared using a transposome-based method where two transposon end sequences, one linked to a tag sequence, and a transposase form a transposome complex. The transposome complexes are used to fragment and tag target nucleic acids in solution
Implementation Method 2
amplifying the plurality of polynucleotides to obtain amplified polynucleotides
Implementation Method 3
enriching for certain sequences in the long fragments with selection probes directed to certain challenging medically relevant genes (CMRG)
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 certain challenging medically relevant genes (CMRG). 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.