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 DNA isolated from a biological sample, wherein the adaptors each comprises a universal priming sequence and a molecular barcode;
performing a first PCR to simultaneously amplify 10 to 20,000 target loci using a first universal primer and 10 to 20,000 target-specific primers in a single reaction volume;
performing a second, nested PCR to simultaneously amplify the 10 to 20,000 target loci using a second universal primer and 10 to 20,000 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 tissue sample.
3 . The method of claim 1 , wherein the method comprises subjecting the isolated DNA to blunting ending, dA-tailing, and adaptor ligation.
4 . The method of claim 1 , wherein the second PCR is one-sided nested PCR.
5 . The method of claim 1 , wherein the method comprises multiplex sequencing of amplified DNA of multiple samples in a single sequencing lane.
6 . 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.
7 . The method of claim 6 , wherein the second PCR comprises simultaneously amplifying between 100 and 5,000 target loci using the second universal primer and between 100 and target-specific primers in a single reaction volume.
8 . The method of claim 1 , wherein the first PCR 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.
9 . The method of claim 8 , 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.
10 . The method of claim 1 , wherein the isolated DNA are tagged with up to 1024 different molecular barcodes.
11 . The method of claim 1 , wherein the isolated DNA are tagged with 1024 −65536 different molecular barcodes.
12 . 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.
13 . 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.
14 . The method of claim 1 , wherein the length of the annealing step of the first and/or second PCR is at least 3 minutes.
15 . The method of claim 1 , wherein the length of the annealing step of the first and/or second PCR is at least 5 minutes.
16 . The method of claim 1 , wherein at least 90% of the amplified DNA map to the target loci.
17 . The method of claim 1 , wherein the target loci are SNP loci.
18 . The method of claim 1 , wherein the DNA comprises DNA from a tumor.
19 . The method of claim 7 , wherein amplified DNAs of multiple samples are pooled and sequenced in a single sequencing lane.