Tm-enhanced blocking oligonucleotides and baits for improved target enrichment and reduced off-target selection
Inventors
Behlke, Mark Aaron • Havens, John Robert • Jarosz, Mirna • Zwirko, Zachary • Lipson, Doron • Juhn, Frank Soo
Assignees
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Publication Number
US-11566282-B2
Publication Date
2023-01-31
Expiration Date
Abstract
The invention is directed to modified oligonucleotide compositions and methods for selectively reducing unwanted nucleic acid contaminants and enriching for desired nucleic acid targets from complex genomic nucleic acid mixtures for sequencing applications. The modified oligonucleotide compositions include one or more modified groups that increase the Tm of the resultant oligonucleotide composition.
Core Innovation
The described invention provides hybrid-capture bait sets and Tm-enhanced oligonucleotides modified with locked nucleic acid or bicyclic nucleic acid groups for selecting desired template nucleic acids from a population. These oligonucleotides serve as blockers and baits that preferentially hybridize to common library adaptor sequences at elevated hybridization stringency. This suppresses adaptor-mediated template concatemer formation, termed daisy chains, and reduces off-target capture and interference from undesired hybridization complexes.
The described methods further encompass massively parallel sequencing of template libraries using specified Tm-enhanced blocker oligonucleotides, bait oligonucleotides, and Cot-1 DNA, followed by isolating and sequencing desired templates. Hybrid complex isolation proceeds via affinity-tag interactions or separation of non-immobilized bait complexes. Disclosed embodiments also utilize individually synthesized DNA oligo-baits to improve sequencing coverage across difficult high-GC targets and apply Bayesian priors to detect rare cancer mutations.
Claims Coverage
The claim coverage includes three inventive features directed to methods for selecting desired template nucleic acids and performing massively parallel sequencing.
Selection using a Tm-enhanced oligonucleotide and affinity-tagged isolation
An independent claim covers contacting a population of template nucleic acids with a Tm-enhanced oligonucleotide containing locked nucleic acid or bicyclic nucleic acid groups selected from specified sequence groups to reduce undesired hybridization complexes, followed by isolating the desired template through hybrid complexes and affinity-tag interaction.
Massively parallel sequencing with Tm-enhanced blockers
An independent claim covers preparing a template-nucleic-acid library and combining it with specified Tm-enhanced blocker oligonucleotides, bait oligonucleotides having substantial sequence complementarity to a desired template sequence, and Cot-1 DNA, followed by isolating and sequencing the desired templates.
Selection using a non-immobilized bait oligonucleotide
An independent claim covers contacting a template-nucleic-acid population with a Tm-enhanced oligonucleotide selected from specified sequence groups and a non-immobilized bait oligonucleotide, forming a hybrid complex between the desired nucleic acid and bait, and separating the complex from the mixture.
Together, the three independent claims cover template-nucleic-acid selection using specified Tm-enhanced oligonucleotides with affinity-tag or non-immobilized bait separation, as well as massively parallel sequencing in the presence of Tm-enhanced blockers and bait oligonucleotides.
Stated Advantages
Tm-enhanced oligonucleotide blockers suppress adaptor-mediated template concatenation, or daisy chains, and reduce off-target capture during hybrid selection.
Extending adaptor-blocker complementarity improves target selection by about 5%.
Target enrichment exceeds 80% with Tm-enhanced blockers compared with about 60% with conventional blockers, achieving enrichment of up to 5,000-fold.
Reported sequencing results demonstrate 99.95% of targeted bases covered above 500x with minimal GC bias.
Individually synthesized DNA oligo-baits improve coverage of difficult high-GC targets, providing approximately four-fold gains with modified bait designs and increased coverage via bait spike-in.
Increased enrichment is achieved with additional hybridization rounds.
Bayesian priors improve the detection of low-frequency, rare cancer mutations.
Documented Applications
Hybrid selection and target enrichment of desired template nucleic acids from a population of template nucleic acids.
Massively parallel sequencing of selected template nucleic acids.
Cancer sequencing and mutation calling, including detection of rare, low-frequency cancer mutations using Bayesian priors.
Bait-set designs for controlling sequencing coverage across target classes, including improving coverage of difficult high-GC targets using individually synthesized DNA oligo-baits.
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