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:
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.