Methods for simultaneous amplification of target loci
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.
1. A method for amplifying and sequencing DNA, comprising:
isolating cell-free DNA from a biological sample and tagging the isolated cell-free DNA, wherein each tagged DNA molecule comprises a molecular barcode;
performing a first PCR to simultaneously amplify at least 10 target loci using a first 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 a second 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; 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 PCR is one-sided nested PCR.
5. The method of claim 1 , wherein the first PCR comprises simultaneously amplifying between 100 and 5,000 target loci using the first universal primer and between 100 and 5,000 target specific primers in a single reaction volume.
6. The method of claim 5 , wherein the second PCR comprises simultaneously amplifying between 100 and 5,000 target loci using the second universal primer and between 100 and 5,000 target-specific primers in a single reaction volume.
7. The method of claim 1 , wherein the first PCR comprises simultaneously amplifying between 1000 and 1,000 target loci using the first universal primer and between 100 and 1,000 inner target-specific primers in a single reaction volume.
8. The method of claim 7 , wherein the second PCR comprises simultaneously amplifying between 100 and 1,000 target loci using the second universal primer and between 100 and 1,000 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 less than 20 nM.
12. The method of claim 1 , wherein each target-specific primer of the first and/or second PCR has a concentration of less than 10 nM.
13. The method of claim 1 , wherein the first and/or second PCR comprises an annealing step having a length of at least 3 minutes.
14. The method of claim 1 , wherein the first and/or second PCR comprises an annealing step having a length of at least 5 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 are SNP 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) by:
i. ligating adaptors to the cfDNA, wherein the adaptors each comprises a universal priming sequence and a molecular barcode;
ii. performing a first amplification to simultaneously amplify at least 10 target loci from the cfDNA using a first universal primer and at least 10 target-specific primers in a single reaction volume;
iii. performing a second, nested amplification to simultaneously amplify the at least 10 target loci using a second 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;
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 1 , wherein the biological sample is a blood, plasma, serum, or urine sample.
24. The method of claim 23 , wherein the method comprises subjecting the cfDNA extracted in (a) to blunting ending, dA-tailing, and adaptor ligation.
25. The method of claim 22 , wherein the second amplification is one-sided nested PCR.
26. 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.
27. The method of claim 22 , wherein the first amplification comprises simultaneously amplifying between 100 and 5,000 target loci using the first universal primer and between 100 and 5,000 target specific primers in a single reaction volume.
28. The method of claim 27 , wherein the second amplification comprises simultaneously amplifying between 100 and 5,000 target loci using the second universal primer and between 100 and 5,000 target-specific primers in a single reaction volume.
29. The method of claim 22 , wherein the first amplification comprises simultaneously amplifying between 100 and 1,000 target loci using the first universal primer and between 100 and 1,000 inner target-specific primers in a single reaction volume.
30. The method of claim 29 , wherein the second amplification comprises simultaneously amplifying between 100 and 1,000 target loci using the second universal primer and between 100 and 1,000 inner target-specific primers in a single reaction volume.
31. The method of claim 22 , wherein the cfDNA extracted in (a) are tagged with up to 1024 different molecular barcodes.
32. The method of claim 22 , wherein the cfDNA extracted in (a) are tagged with 1024-65536 different molecular barcodes.
33. The method of claim 22 , wherein each target-specific primer of the first and/or second amplification has a concentration of less than 20 nM.
34. The method of claim 22 , wherein each target-specific primer of the first and/or second amplification has a concentration of less than 10 nM.
35. The method of claim 22 , wherein the first and/or second amplification comprises an annealing step having a length of at least 3 minutes.
36. The method of claim 22 , wherein the first and/or second amplification comprises an annealing step having a length of at least 5 minutes.
37. The method of claim 22 , wherein at least 90% of the enriched fraction of the cfDNA map to the target loci.
38. The method of claim 22 , wherein the target loci are SNP loci.
39. The method of claim 22 , wherein the cfDNA extracted in (a) comprises DNA from mixed origin.
40. The method of claim 22 , wherein the cfDNA extracted in (a) comprises DNA from a patient suspected of having cancer.
41. The method of claim 39 , wherein the cfDNA extracted in (a) comprises DNA from a tumor.
42. The method of claim 39 , wherein the cfDNA extracted in (a) comprises DNA from a transplant.
43. The method of claim 28 , wherein the enriched fraction of the cfDNA from multiple samples are pooled and sequenced in a single sequencing lane.