Methods for determining a nucleotide sequence contiguous to a known target nucleotide sequence
Inventors
Iafrate, Anthony John • Le, Long Phi • Zheng, Zongli
Assignees
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Publication Number
US-10718009-B2
Publication Date
2020-07-21
Expiration Date
Abstract
The technology described herein is directed to methods of determining oligonucleotide sequences, e.g. by enriching target sequences prior to sequencing the sequences.
Core Innovation
The disclosure relates to determining the nucleotide sequence contiguous to a known target nucleotide sequence. It provides a method in which a target nucleic acid comprising the known target nucleotide sequence is ligated with a universal oligonucleotide tail-adaptor comprising a blocking strand and an amplification strand, with a first ligatable duplex end and a second unpaired end.
After ligation, the method amplifies a portion of the target nucleic acid and the amplification strand with a first adaptor primer and a first target-specific primer, and then amplifies a portion of the resulting amplicon with a second adaptor primer and a second target-specific primer. The second target-specific primer is nested with respect to the first target-specific primer, and the universal adaptor primers are constrained by nested relationships and sequence identity to sequencing primer portions.
The amplified portion resulting from the second amplification is sequenced using first and second sequencing primers. The amplification strand comprises nucleic acid sequences identical to the sequencing primers, and the adaptor and amplification strand structure supports sequencing of the region contiguous to the known target.
Claims Coverage
Independent claim coverage is provided for one complete workflow that ligates a universal oligonucleotide tail-adaptor, performs two rounds of nested adaptor/target-specific amplification, and sequences the amplified portion. The inventive features in the claim are directed to the structure and relationships of the universal adaptor and primers, and to sequencing of nucleotide sequence contiguous to a known target.
Universal oligonucleotide tail-adaptor with blocking and amplification strands
The method ligates a target nucleic acid comprising the known target nucleotide sequence with a universal oligonucleotide tail-adaptor, wherein the universal oligonucleotide tail-adaptor comprises a blocking strand and an amplification strand, and wherein the universal oligonucleotide tail-adaptor comprises a first ligatable duplex end and a second unpaired end.
Adaptor/target two-round amplification with nested target-specific primers
The method amplifies a portion of the target nucleic acid and the amplification strand with a first adaptor primer and a first target-specific primer, and amplifies a portion of the amplicon with a second adaptor primer and a second target-specific primer, wherein the second target-specific primer is nested with respect to the first target-specific primer.
Sequencing using sequencing primers defined by adaptor and amplification strand sequence identity
The method sequences the amplified portion from the second amplification using a first and second sequencing primer, wherein the amplification strand comprises nucleic acid sequences identical to the first and second sequencing primers, and the adaptor primers comprise sequence identity to sequencing primer portions.
The independent claim covers determining nucleotide sequence contiguous to a known target by ligating a universal tail-adaptor having blocking and amplification strands with specified duplex and unpaired structure, performing two rounds of adaptor/target-specific amplification using nested target-specific primers, and sequencing with sequencing primers enabled by adaptor and amplification-strand sequence identity.
Stated Advantages
High on-target/on-target rate.
Reduced primer-dimer carryover using tagged primers and half-truncated “Y” adapters.
Documented Applications
Detecting gene rearrangements/oncogenic fusion partners in which only one known breakpoint flank is used for contiguity determination.
Clinical cancer treatment and treatment-responsiveness decision methods based on detected oncogene rearrangements, including ALK, ROS1, and RET.
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