Granted Patent B2

US 12,668,843 · App. 18/620,822 · Granted Jun 30, 2026

Inventors: Matthew Rabinowitz (San Francisco, CA); Matthew Micah Hill (Belmont, CA); Bernhard Zimmermann (Manteca, CA); Johan Baner (San Francisco, CA); George Gemelos (Portland, OR); Milena Banjevic (Los Altos Hills, CA); Allison Ryan (Belmont, CA); Styrmir Sigurjonsson (San Jose, CA); Zachary Demko (San Francisco, CA)

Assignee: Natera, Inc.

C12Q1/6883C12Q1/6809C12Q1/6811C12Q1/6844C12Q1/6848C12Q1/6851C12Q1/6855C12Q1/6869C12Q1/6874C12Q2600/156

Abstract

Claims (16)

1 . A method for preparing tagged amplified products useful for analyzing a plurality of polymorphic loci associated with cancer, comprising

performing targeted multiplex amplification on cell-free DNA extracted from a plasma sample of a human subject to obtain a plurality of amplicons, wherein the targeted multiplex amplification amplifies 20-2,000 polymorphic loci associated with cancer in a single reaction volume;

ligating the plurality of amplicons with one or more universal adaptors and a plurality of different molecular barcodes to obtain tagged amplicons;

amplifying the tagged amplicons using the universal adaptors to generate tagged amplified products each containing at least one polymorphic locus associated with cancer, a molecular barcode, and one or more sequencing tags; and

conducting massively parallel sequencing on the tagged amplified products to produce sequence reads.

2 . The method of claim 1 wherein the polymorphic loci comprise single nucleotide polymorphisms or single nucleotide variants or both.

3 . The method of claim 1 , wherein the one or more universal tail adaptors each comprise a first strand and a second strand, wherein a first end of each of the universal tail adaptors comprises a double-stranded section comprising the 5′ portion of the first strand and the 3′ portion of the second strand, wherein the first end is ligated to the nucleic acids.

4 . The method of claim 3 , wherein the targeted multiplex amplification comprises a first amplifying step and a second amplifying step, wherein the first amplifying step comprises using a first target-specific primer that anneals to a specific target sequence and a first adaptor primer having a nucleotide sequence identical to a first portion of the first strand to generate a first amplification product.

5 . The method of claim 4 , wherein the second amplifying step comprises using a second target-specific primer that anneals to the first amplification product and a second adaptor primer having a nucleotide sequence identical to a second portion of the first strand to generate the final amplification product.

6 . The method of claim 5 , wherein the second adaptor primer is nested relative to the first adaptor primer.

7 . The method of claim 6 , wherein the second target-specific primer comprises an index tag.

8 . The method of claim 7 , wherein the second amplifying step further comprises using an index primer comprising a sequence complementary to the index tag.

9 . The method of claim 1 , wherein the one or more universal tail adaptors comprise a second sequencing tag.

10 . The method of claim 1 , wherein the one or more universal tail adaptors comprise a first universal tail adaptor and a second universal tail adaptor.

11 . The method of claim 1 , wherein the targeted multiplex amplification comprises a single amplifying step.

12 . The method of claim 11 , wherein the targeted multiplex amplification comprises using a third primer and a fourth primer, wherein the third primer comprises a first sequencing tag and wherein the fourth primer comprises a second sequencing tag.

Assignments (2)

Continuity (40)

References Cited (400)