Methods for simultaneous amplification of target loci

• US 10,538,814 B2
• Filed: 04/30/2019
• Issued: 01/21/2020
• Est. Priority Date: 05/18/2010
• Status: Active Grant

• Current Assignee: Natera Incorporated (Natera)
• Original Assignee: Natera Incorporated (Natera)
• Inventors: Babiarz, Joshua, Constantin, Tudor Pompiliu, Eubank, Lane A., Gemelos, George, Hill, Matthew Micah, Kirkizlar, Huseyin Eser, Rabinowitz, Matthew, Sakarya, Onur, Sigurjonsson, Styrmir, Zimmermann, Bernhard
• Primary Examiner(s): Mummert, Stephanie K
• Application Number:
  US16/399,268
• Publication Number:
  US 20190256917A1
• Time in Patent Office: 266 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.

Claims

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

ligating adaptors to cell-free DNA isolated from a biological sample, wherein the adaptors each comprises a universal priming site;

performing a first PCR to simultaneously amplify at least 10 target loci using a universal primer and at least 10 target-specific primers in a single reaction volume;

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

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.

4. The method of claim 1, wherein the adaptor further comprises a molecular barcode.

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

6. The method of claim 1, wherein the method comprises multiplex sequencing of amplified DNA of multiple samples in a single sequencing lane.

7. The method of claim 1, wherein the first PCR comprises simultaneously amplifying at least 50 target loci using the universal primer and at least 50 target-specific primers in a single reaction volume.

8. The method of claim 1, wherein the first PCR comprises simultaneously amplifying at least 100 target loci using the universal primer and at least 100 target-specific primers in a single reaction volume.

9. The method of claim 1, wherein the second PCR comprises simultaneously amplifying at least 50 target loci using the universal primer and at least 50 inner target-specific primers in a single reaction volume.

10. The method of claim 1, wherein the second PCR comprises simultaneously amplifying at least 100 target loci using the universal primer and at least 100 inner target-specific primers in a single reaction volume.

11. The method of claim 4, wherein the isolated cell-free DNA are tagged with up to 1024 different molecular barcodes.

12. The method of claim 4, wherein the isolated cell-free DNA are tagged with 1024-65536 different molecular barcodes.

13. The method of claim 1, wherein the concentration of each target-specific primer of the first and/or second PCR is less than 20 nM.

14. The method of claim 1, wherein the concentration of each target-specific primer of the first and/or second PCR is less than 10 nM.

15. The method of claim 1, wherein the length of the annealing step of the first and/or second PCR is at least 3 minutes.

16. The method of claim 1, wherein the length of the annealing step of the first and/or second PCR is at least 5 minutes.

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

18. The method of claim 1, wherein the target loci are SNP loci.

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

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

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

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

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

ligating adaptors to cell-free DNA isolated from a biological sample, wherein the adaptors each comprises a universal priming site, wherein the cell-free DNA comprises DNA from mixed origin;

performing a first PCR to simultaneously amplify at least 100 target loci using a universal primer and at least 100 target-specific primers in a single reaction volume;

performing a second, one-sided nested PCR to simultaneously amplify the at least 100 target loci using the universal primer and at least 100 inner target-specific primers in a single reaction volume, wherein at least one of the primers comprises a sequencing tag;

performing high-throughput sequencing to sequence the amplified DNA comprising the target loci, wherein the target loci are SNP loci, and wherein amplified DNAs of multiple samples are pooled and sequenced in a single sequencing lane.