Granted Patent B2

US 12,545,960 · App. 19/028,873 · Granted Feb 10, 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 (38)

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

isolating cell-free DNA from a biological sample and tagging the isolated cell-free DNA to obtain tagged DNA, wherein each tagged DNA molecule comprises a universal priming sequence and a molecular barcode;

performing universal amplification on the tagged DNA;

performing a first targeted PCR to simultaneously amplify 10 to 1,000 target loci using a first universal primer and 10 to 1,000 target-specific primers in a single reaction volume;

performing a second targeted, nested PCR to simultaneously amplify the 10 to 1,000 target loci using a second universal primer and 10 to 1,000 inner target-specific primers in a single reaction volume, wherein at least one of the primers comprises a sequencing tag; and

performing high-throughput sequencing to sequence the amplified DNA comprising the target loci.

2 . The method of claim 1 , wherein the biological sample is a blood, plasma, serum, or urine sample.

3 . The method of claim 1 , wherein the method comprises subjecting the isolated cell-free DNA to blunting ending, dA-tailing, and adaptor ligation, wherein the adaptor comprises a universal priming site and the molecular barcode.

4 . The method of claim 1 , wherein the second targeted PCR is one-sided nested PCR.

5 . The method of claim 1 , wherein the first targeted PCR comprises simultaneously amplifying 20 to 500 target loci using the first universal primer and 20 to 500 target specific primers in a single reaction volume.

6 . The method of claim 5 , wherein the second targeted PCR comprises simultaneously amplifying 20 to 500 target loci using the second universal primer and 20 to 500 target-specific primers in a single reaction volume.

7 . The method of claim 1 , wherein the first targeted PCR comprises simultaneously amplifying 20 to 200 target loci using the first universal primer and 20 to 200 inner target-specific primers in a single reaction volume.

8 . The method of claim 6 , wherein the second targeted PCR comprises simultaneously amplifying 20 to 200 target loci using the second universal primer and 20 to 200 inner target-specific primers in a single reaction volume.

9 . The method of claim 1 , wherein the isolated cell-free DNA are tagged with up to 1024 different molecular barcodes.

10 . The method of claim 1 , wherein the isolated cell-free DNA are tagged with 1024-65536 different molecular barcodes.

11 . The method of claim 1 , wherein each target-specific primer of the first and/or second PCR has a concentration of 1-50 nM.

12 . The method of claim 1 , wherein each target-specific primer of the first and/or second PCR has a concentration of 1-20 nM.

13 . The method of claim 1 , wherein the first and/or second PCR comprises an annealing step having a length of longer than 2 minutes.

14 . The method of claim 1 , wherein the first and/or second PCR comprises an annealing step having a length of longer than 4 minutes.

15 . The method of claim 1 , wherein at least 90% of the amplified DNA map to the target loci.

16 . The method of claim 1 , wherein the target loci comprise single nucleotide polymorphism or variant loci.

17 . The method of claim 1 , wherein the cell-free DNA comprises DNA from mixed origin.

18 . The method of claim 17 , wherein the cell-free DNA comprises DNA from a fetus.

19 . The method of claim 17 , wherein the cell-free DNA comprises DNA from a tumor.

20 . The method of claim 17 , wherein the cell-free DNA comprises DNA from a transplant.

21 . The method of claim 7 , wherein amplified DNAs of multiple samples are pooled and sequenced in a single sequencing lane.

22 . A method for preparing an enriched deoxyribonucleic acid (DNA) fraction of cell-free DNA (cfDNA) useful for analyzing target loci, comprising:

(a) extracting cfDNA from a biological sample;

(b) producing an enriched fraction of the cfDNA extracted in (a), comprising: (i) tagging the cfDNA to obtain tagged DNA, wherein each tagged DNA molecule comprises a universal priming sequence and a molecular barcode; (ii) performing universal amplification on the tagged DNA; (iii) performing a first targeted PCR to simultaneously amplify 10 to 1,000 target loci using a first universal primer and 10 to 1,000 target-specific primers in a single reaction volume; (iv) performing a second targeted, nested PCR to simultaneously amplify the 10 to 1,000 target loci using a second universal primer and 10 to 1,000 inner target-specific primers in a single reaction volume, wherein at least one of the primers comprises a sequencing tag;

(c) analyzing the target loci by performing high-throughput sequencing to sequence the enriched fraction of the cfDNA comprising the target loci.

23 . The method of claim 22 , wherein the biological sample is a blood, plasma, serum, or urine sample, wherein the target loci comprise single nucleotide polymorphism or variant loci.

24 . The method of claim 22 , wherein the method comprises multiplex sequencing of the enriched fraction of the cfDNA from multiple samples in a single sequencing lane.

25 . The method of claim 22 , wherein the first targeted PCR comprises simultaneously amplifying 20 to 500 target loci using the first universal primer and 20 to 500 target specific primers in a single reaction volume.

26 . The method of claim 25 , wherein the second targeted PCR comprises simultaneously amplifying 20 to 500 target loci using the second universal primer and 20 to 500 target-specific primers in a single reaction volume.

27 . The method of claim 22 , wherein the first targeted PCR comprises simultaneously amplifying 20 to 200 target loci using the first universal primer and 20 to 200 inner target-specific primers in a single reaction volume.

28 . The method of claim 27 , wherein the second targeted PCR comprises simultaneously amplifying 20 to 200 target loci using the second universal primer and 20 to 200 inner target-specific primers in a single reaction volume.

29 . The method of claim 22 , wherein the cfDNA extracted in (a) are tagged with up to 1024 different molecular barcodes.

30 . The method of claim 22 , wherein the cfDNA extracted in (a) are tagged with 1024-65536 different molecular barcodes.

Continuity (41)

References Cited (400)