Granted Patent B2

US 12,692,544 · App. 19/028,937 · Granted Jul 28, 2026

Inventors: Matthew Rabinowitz (San Francisco, CA); Matthew Hill (Belmont, CA); Bernhard Zimmermann (Manteca, CA); George Gemelos (Portland, OR); Johan Baner (San Francisco, CA); 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 (19)

1 . A method for amplifying and sequencing DNA, comprising:

extracting cell-free DNA from a blood sample or a portion thereof collected from a subject who is not a pregnant woman;

amplifying extracted cell-free DNA or DNA derived therefrom one or more times to generate amplification products, wherein one of the amplification steps comprises targeted amplification of a plurality of single nucleotide polymorphism or variant loci in a single reaction volume, wherein the plurality of single nucleotide polymorphism or variant loci comprises 25-2,000 loci associated with cancer, wherein the method does not comprise splitting the extracted cell-free DNA or DNA derived therefrom into parallel reactions and pooling of amplification products from parallel reactions;

tagging the amplification products with one or more universal tail adaptors to generate tagged products, and amplifying the tagged products using adaptor-specific primers to generate final amplification products, wherein the final amplification products comprise at least one barcode and at least one sequencing tag, wherein tagging the amplification products comprises appending the one or more universal tail adaptors to the amplification products; and

performing massively parallel sequencing on the final amplification products to sequence the plurality of single nucleotide polymorphism or variant loci, wherein the final amplification products derived from multiple samples are combined and sequenced together.

2 . The method of claim 1 , wherein the method does not comprise targeted amplification on microfluidic chips.

3 . The method of claim 1 , wherein the method does not comprise targeted amplification by droplet PCR.

4 . The method of claim 1 , wherein tagging the amplification products comprises ligating the one or more universal tail adaptors to the amplification products.

5 . The method of claim 4 , 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 amplification products.

6 . The method of claim 1 , wherein the one or more universal tail adaptors comprise the barcode.

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

8 . 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.

9 . The method of claim 1 , wherein amplifying the tagged products comprises a single amplifying step.

10 . The method of claim 1 , wherein amplifying the tagged products comprises using a first adaptor-specific primer and a second adaptor-specific primer, wherein the first adaptor-specific primer comprises a first sequencing tag and wherein the second adaptor-specific primer comprises a second sequencing tag.

11 . The method of claim 1 , wherein at least one amplicon in the amplification products comprises two or more nearby single nucleotide polymorphism or variant loci.

12 . The method of claim 1 , wherein the cell-free DNA is extracted from a plasma portion of the blood sample.

13 . The method of claim 1 , wherein the massively parallel sequencing is performed with an average depth of read of at least 400.

14 . The method of claim 1 , wherein the targeted amplification comprises amplifying 50-2,000 single nucleotide polymorphism or variant loci in a single reaction volume.

15 . The method of claim 1 , wherein the targeted amplification comprises amplifying 100-1,000 single nucleotide polymorphism or variant loci in a single reaction volume.

Assignments (2)

Continuity (41)

References Cited (400)