Systems and methods for allele enrichment using multiplexed blocker displacement amplification
Inventors
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Rice UniversityRice University is a leading research university in Houston, Texas, recognized for its emphasis on scientific discovery, innovation, and interdisciplinary collaboration. The institution is committed to academic excellence, impactful research, and community engagement, offering robust undergraduate and graduate programs in engineering, natural sciences, social sciences, humanities, business, and the arts. Rice is distinguished by its history of collaboration with organizations such as NASA, fostering advances in space science, biotechnology, energy research, and artificial intelligence.
Rice University is a leading research university in Houston, Texas, recognized for its emphasis on scientific discovery, innovation, and interdisciplinary collaboration. The institution is committed to academic excellence, impactful research, and community engagement, offering robust undergraduate and graduate programs in engineering, natural sciences, social sciences, humanities, business, and the arts. Rice is distinguished by its history of collaboration with organizations such as NASA, fostering advances in space science, biotechnology, energy research, and artificial intelligence.
Publication Number
US-12331350-B2
Publication Date
2025-06-17
Expiration Date
Abstract
Provided herein are reagents and methods for simultaneously enriching many potential rare genetic variants at different genetic loci. The rare variants enriched can include single nucleotide polymorphisms (SNPs), single nucleotide variants, or small insertions and deletions. Embodiments of the invention include procedures for integration with downstream next generation sequencing (NGS) analysis. Embodiments of the invention include analysis of nonpathogenic SNPs for the determination of cell identity and detection of cell contamination using qPCR or NGS.
Core Innovation
Provided herein are reagents and methods for simultaneously enriching many potential rare genetic variants at different genetic loci. The rare variants enriched can include single nucleotide polymorphisms (SNPs), single nucleotide variants, or small insertions and deletions. Embodiments of the invention include procedures for integration with downstream next generation sequencing (NGS) analysis and analysis of nonpathogenic SNPs for the determination of cell identity and detection of cell contamination using qPCR or NGS.
Sequence variations in genomic DNA include nonpathogenic single nucleotide polymorphisms (SNPs) that can collectively distinguish individuals from each other, pathogenic germline mutations that can cause or increase the likelihood of genetic diseases, and pathogenic somatic mutations that cause cancer. The technical difficulty distinguishing these sequence variations depends strongly on both the fraction of the DNA that contains the variation (the variant allele fraction; VAF) and the number of variations that need to be simultaneously profiled. Simultaneously profiling many sequence variations each at potentially low VAF remains a significant challenge because microarrays lack the sensitivity for low VAFs, digital PCR cannot be multiplexed past a very small number, and ultradeep NGS is slow and cost prohibitive when applied to many potential mutations.
Provided herein are reagents and methods to simultaneously enrich many different sequence variations having low VAFs, enabling profiling via low-depth NGS or microarrays in highly multiplexed settings. In one embodiment, the methods use blocker displacement amplification (BDA) oligo sets for multiple genetic loci, each BDA oligo set comprising a BDA forward primer, a BDA blocker, and a BDA reverse primer, wherein several 3′-most nucleotides of the forward primer are also present at or near the 5′ end of the blocker and wherein each blocker contains a 3′ sequence or modification that prevents extension by DNA polymerase; the mixture is then amplified and the resulting amplicons are analyzed by NGS. These methods are described as applicable to multiplex enrichment across many loci and for applications including detection of cell line contamination and analysis of rare cancer mutations in liquid biopsy settings.
Claims Coverage
Overview: One independent claim defines seven inventive features.
Simultaneous amplification at ten or more loci
A method for simultaneously amplifying and detecting allelic variants at at least ten genetic loci.
BDA oligo set composition
Each genetic locus is targeted with a blocker displacement amplification (BDA) oligo set comprising a BDA forward primer, a BDA blocker, and a BDA reverse primer.
Forward primer–blocker overlap requirement
At least four nucleotides at the 3′ end of each BDA forward primer sequence are also present at or near the 5′ end of its respective BDA blocker sequence.
Blocker 3′ extension-preventing modification
Each BDA blocker contains a 3′ sequence or modification that prevents extension by DNA polymerase.
Blocker concentration relative to forward primer
The concentration of each BDA blocker is required to be greater than its respective BDA forward primer [procedural detail omitted for safety].
Amplification to produce amplicons
The mixed sample, DNA polymerase, and BDA oligo sets are subjected to amplification cycles to produce amplicons [procedural detail omitted for safety].
Next-generation sequencing of amplicons
Performing next-generation sequencing (NGS) of the produced amplicons.
The independent claim covers a multiplexed BDA workflow that uses specified BDA oligo set composition and primer–blocker overlap, requires blockers with 3′ extension-preventing modifications and higher blocker concentration relative to forward primers, involves amplifying the mixture to produce amplicons, and performing NGS of the amplicons.
Stated Advantages
Simultaneous enrichment of many different sequence variations having low VAFs, enabling profiling via low-depth NGS or microarrays.
Enrichment of rare variants by large fold changes (described as by 100-fold or more) allowing sensitive detection of rare sequence variants.
Integration with downstream NGS analysis to enable detection and quantitation of rare variants while reducing required sequencing reads compared to standard NGS and UMI-based methods, thereby reducing cost.
Applications for detection of cell line contamination and analysis of rare cancer mutations in liquid biopsy settings, and for determination or verification of genomic identity using panels of nonpathogenic SNPs.
Documented Applications
Detection of cell line contamination using qPCR or NGS.
Analysis of rare cancer mutations in liquid biopsy settings (noninvasive cancer profiling via cell-free DNA in plasma).
Integration with downstream next generation sequencing (NGS) analysis of enriched amplicons.
Determination or verification of cell identity or genomic identity of an individual or organism using panels of nonpathogenic SNPs.
Calculation of a quantitative estimate of the fraction of a minority cell type from a heterogeneous cell sample by taking a median or mean of inferred VAF values for three or more different variants.
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