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 cell-free DNA, comprising:
performing a multiplex targeted pre-amplification of cell-free DNA isolated from a biological sample, wherein the pre-amplification is a linear amplification;
performing a first PCR to simultaneously amplify at least 10 target loci in one reaction volume using a first universal primer and at least 10 target-specific primers;
performing a second, nested PCR to simultaneously amplify the at least 10 target loci in one reaction volume using a second universal primer and at least 10 inner target-specific primers; and
performing high-throughput sequencing to sequence the amplified target loci and determine whether the target loci comprise a cancer-associated mutation, wherein the target loci are single nucleotide variant (SNV) 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 first PCR comprises simultaneously amplifying at least 20 target loci in one reaction volume.
4. The method of claim 1 , wherein the first PCR comprises simultaneously amplifying at least 50 target loci in one reaction volume.
5. The method of claim 1 , wherein the first PCR comprises simultaneously amplifying at least 100 target loci in one reaction volume.
6. The method of claim 1 , wherein the second PCR comprises simultaneously amplifying at least 20 target loci in one reaction volume.
7. The method of claim 1 , wherein the second PCR comprises simultaneously amplifying at least 50 target loci in one reaction volume.
8. The method of claim 1 , wherein the second PCR comprises simultaneously amplifying at least 100 target loci in one reaction volume.
9. The method of claim 1 , wherein the amplified target loci are tagged with molecular barcodes.
10. The method of claim 1 , wherein the amplified target loci are tagged with up to 1024 molecular barcodes.
11. The method of claim 1 , wherein the amplified target loci are tagged with 1024-65536 molecular barcodes.
12. The method of claim 1 , wherein at least one of the target specific primers comprises a tail, wherein the tail has no homology to the target loci and comprises a common priming site.
13. The method of claim 1 , wherein at least one of the target specific primers comprises a priming site for a subsequent amplification to add barcode sequences for multiplex sequencing.
14. The method of claim 1 , wherein the method further comprises barcoding PCR to introduce a sample-specific barcode, and wherein amplified DNAs from multiple samples are pooled together and sequenced in a single sequencing lane.
15. A method for amplifying and sequencing cell-free DNA, comprising:
performing a multiplex targeted pre-amplification of cell-free DNA isolated from a biological sample, wherein the pre-amplification is a linear amplification;
performing a multiplex targeted nested PCR to simultaneously amplify at least 10 target loci in one reaction volume; and
performing high-throughput sequencing to sequence the amplified target loci and determine whether the target loci comprise a cancer-associated mutation, wherein the target loci are single nucleotide variant (SNV) loci.
16. The method of claim 15 , wherein the multiplex targeted nested PCR comprises one-sided nested PCR.
17. The method of claim 15 , wherein the multiplex targeted nested PCR simultaneously amplifies least 20 target loci in one reaction volume.
18. The method of claim 15 , wherein the multiplex targeted nested PCR simultaneously amplifies at least 50 target loci in one reaction volume.
19. The method of claim 15 , wherein the amplified target loci are tagged with molecular barcodes.
20. The method of claim 15 , wherein the method further comprises barcoding PCR to introduce a sample-specific barcode, and wherein amplified DNAs of multiple samples are pooled together and sequenced in a single sequencing lane.