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 at least one adaptor to cell-free DNA isolated from a biological sample or DNA derived therefrom, wherein the adaptor comprise a universal priming site;
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 on the amplified DNA to obtain sequence reads, wherein the target loci are single nucleotide polymorphism or variant 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 adaptor comprises a molecular barcode.
10 . The method of claim 1 , wherein at least 80% of the amplified DNA maps to the target loci.
11 . 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.
12 . 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.
13 . The method of claim 1 , wherein the method further comprises barcoding PCR to introduce a sample-specific barcode, and wherein the amplified DNA from multiple samples are pooled together and sequenced in a single sequencing lane.
14 . The method of claim 1 , wherein the method further comprises determining the presence of cancer DNA based on the sequence reads.
15 . A method for amplifying and sequencing DNA, comprising:
ligating at least one adaptor to cell-free DNA isolated from a biological sample or DNA derived therefrom, wherein the adaptor comprise a universal priming site;
performing a multiplex targeted nested PCR to simultaneously amplify at least 10 target loci in one reaction volume, wherein the multiplex targeted nested PCR comprises semi-nested PCR, hemi-nested PCR, one-sided nested PCR, or fully nested PCR; and
performing high-throughput sequencing on the amplified DNA to obtain sequence reads and determine the presence of cancer DNA based on the sequence reads, wherein the target loci are single nucleotide polymorphism or variant loci.
16 . The method of claim 15 , wherein the biological sample is a blood, plasma, serum, or urine sample.
17 . The method of claim 15 , wherein the multiplex targeted nested PCR simultaneously amplifies least 20 target loci in one reaction volume, and wherein at least 80% of the sequence reads map to the target loci.
18 . The method of claim 15 , wherein the multiplex targeted nested PCR simultaneously amplifies at least 50 target loci in one reaction volume, and wherein at least 90% of the sequence reads map to the target loci.
19 . The method of claim 15 , wherein the adaptor comprises a molecular barcode.
20 . The method of claim 15 , wherein the method further comprises barcoding PCR to introduce a sample-specific barcode, and wherein the amplified DNA of multiple samples are pooled together and sequenced in a single sequencing lane.