Long-Read Nucleic Acid Libraries for Low-Mappability Enrichment
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Solution Overview
Problem
Current nucleic acid library preparation methods for next-generation sequencing are inefficient, costly, and difficult due to the need for multiple steps, material transfers, and expensive instruments, and they often underrepresent certain genomic regions.
Innovation Solution
A method involving immobilized transposomes on a solid support, such as beads, to generate and amplify long nucleic acid fragments, followed by mutagenesis PCR and suppression PCR to enrich for target sequences, and adding library adapters, while using low bias DNA polymerases and nucleotide analogues to introduce mutations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If transposome-based methods are used to fragment and tag target nucleic acids, then library preparation time and cost are reduced, but certain portions of the genome become underrepresented in the libraries
Solution Approach 1:
The invention divides the genome into two categories: mappable regions and low-mappability regions. By creating separate probe sets for each category, the method selectively enriches for low-mappability regions that would otherwise be underrepresented in standard transposome-based libraries, while maintaining overall library preparation efficiency
Solution Approach 2:
The invention changes the selection parameter by introducing selection probes that specifically target low-mappability genomic regions. This parameter change allows selective enrichment of previously underrepresented sequences, improving genome representation uniformity without sacrificing the productivity gains of transposome-based methods
2Reliability
If multiple steps and material transfers are employed in library preparation, then sequencing quality is improved, but hands-on time and operational complexity increase
Solution Approach 1:
The invention merges the enrichment step with the library preparation workflow by using selection probes that can be incorporated into the existing transposome-based protocol. This integration maintains sequencing quality through selective enrichment while avoiding the need for separate, time-consuming enrichment procedures
Solution Approach 2:
The selection probes are designed to automatically enrich for low-mappability regions during the library preparation process itself, without requiring additional manual intervention or separate enrichment steps. The system performs the enrichment function as part of its standard operation, reducing hands-on time while maintaining quality
3Productivity
If standard library preparation protocols are used, then sequencing coverage is achieved, but underrepresented genomic regions remain poorly covered
Solution Approach 1:
The invention introduces selection probes as an intermediary element that mediates between the transposome-based library preparation and the final sequencing outcome. These probes selectively bind to low-mappability regions, ensuring they are properly represented in the final library without disrupting the overall productivity of the sequencing workflow
Solution Approach 2:
By changing the selection parameter to specifically target low-mappability regions, the invention improves genomic region representation while maintaining the productivity of standard library preparation protocols. The parameter change enables selective enrichment without requiring a complete protocol overhaul
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 hands-on time and reagent requirements, allows the use of unpurified samples, and enhances the representation of underrepresented genomic regions, providing efficient and cost-effective nucleic acid library preparation.
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 to generate a sequencer-ready tagmented library
Implementation Method 2
amplifying the plurality of polynucleotides to obtain amplified polynucleotides
Implementation Method 3
using low bias DNA polymerases and nucleotide analogues to introduce mutations
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 genes throughout the genome and expressed regions with low mappability. 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.