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-11781179-B2
Publication Date
2023-10-10
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 invention provides a method for determining a nucleotide sequence contiguous to a known target nucleotide sequence. A nucleic acid template comprising a 3′-end with a known target nucleotide sequence and a first nucleotide sequence contiguous to the known target nucleotide sequence is amplified to generate amplification products containing the first nucleotide sequence and the known target nucleotide sequence. The method uses nested amplification to generate a second amplification product and then determines the sequence of the first nucleotide sequence contained in the second amplification product.
In one approach, an amplification strand of a universal oligonucleotide tail-adaptor is ligated to a 5′-end of the nucleic acid template. The ligated universal oligonucleotide tail-adaptor includes an amplification strand used for primer-based amplification, where the first primer comprises a nucleotide sequence identical to a 5′ portion of a first sequencing primer and the third primer comprises a nucleotide sequence identical to a portion of the first sequencing primer and is nested relative to the first primer. Sequencing of the second amplification product uses sequencing primers compatible with next-generation sequencing, including sequencing primer portions reflected in the amplification primers.
The universal oligonucleotide tail-adaptor is described as including a blocking strand and an amplification strand with a ligatable duplex end and a 3′ T overhang, and optionally as a Y-shaped adapter including a barcode portion. The workflow is further described as improving specificity using a half-truncated Y adapter reducing carryover background and using nested/unidirectional primer design. Tagged and/or structured primers are described as suppressing primer dimer background, supporting a sequencing workflow to determine contiguous sequence without needing both fusion partners.
Claims Coverage
The document provides two independent claims. Both claims cover a nested amplification workflow that uses sequencing-primer-compatible primers, and then determines the sequence of the first nucleotide sequence in a second amplification product; one claim additionally requires ligation of a universal oligonucleotide tail-adaptor and next-generation sequencing as the determining step.
Nested amplification to determine contiguous nucleotide sequence
A method in which a nucleic acid template comprising a 3′-end with a known target nucleotide sequence and a first nucleotide sequence contiguous thereto is amplified using a first primer and a second primer to generate a first amplification product, then the first amplification product is amplified using a third primer nested relative to the first primer and a fourth primer nested relative to the second primer to generate a second amplification product, and the sequence of the first nucleotide sequence in the second amplification product is determined.
Universal oligonucleotide tail-adaptor ligation and next-generation sequencing readout
A method in which an amplification strand of a universal oligonucleotide tail-adaptor is ligated to a 5′-end of a nucleic acid template comprising a 3′-end with a known target nucleotide sequence and a first nucleotide sequence contiguous thereto, a first amplification product is generated using primers that comprise nucleotide sequences identical to sequencing primer portions while targeting the known target sequence, a second amplification product comprising the first nucleotide sequence is generated using nested primers, and the sequence of the first nucleotide sequence is determined by a process comprising next-generation sequencing.
Collectively, the independent claims define nested amplification strategies using primers that include nucleotide sequences identical to sequencing primer portions, with sequencing of the first nucleotide sequence contained in the second amplification product; the second independent claim further anchors the workflow on ligation of a universal oligonucleotide tail-adaptor and determining the sequence via next-generation sequencing.
Stated Advantages
Increased specificity for determining contiguous sequence while reducing carryover background.
Improved background suppression, including suppression of primer dimers.
Enables determining nucleotide sequence contiguous to a known target sequence without needing both fusion partners.
Documented Applications
Detecting gene rearrangements and fusion oncogenes (ALK/ROS1/RET) from genomic DNA or RNA/FFPE-derived material.
Cancer treatment selection and response assessment in relation to indicated inhibitors (e.g., ALK inhibitor, ROS1 inhibitor, RET inhibitor; example includes crizotinib for ALK/ROS1).
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