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 comprising:
(a) performing multiplex targeted pre-amplification of at least 10 target loci from cell-free DNA isolated from a biological sample in a single reaction volume using at least 10 different target-specific primers to obtain amplified DNA, wherein the biological sample is a blood, plasma, serum, or urine sample;
(b) tagging the amplified DNA with a plurality of different molecular barcodes and a sequencing adapter to obtain molecularly barcoded amplified DNA; and
(c) performing high-throughput sequencing of the molecularly barcoded amplified DNA to sequence the at least 10 amplified target loci to generate sequence reads with a depth of at least 200 reads per target locus, wherein the target loci comprises a cancer-associated mutation, and wherein the target loci are single nucleotide variant (SNV) loci.
2 . The method of claim 1 , wherein at least 20 target loci are pre-amplified in a single reaction volume using at least 20 different target-specific primers to obtain amplified DNA.
3 . The method of claim 1 , wherein at least 50 target loci are pre-amplified in a single reaction volume using at least 50 different target-specific primers to obtain amplified DNA.
4 . The method of claim 1 , wherein at least 100 target loci are pre-amplified in a single reaction volume using at least 100 different target-specific primers to obtain amplified DNA.
5 . The method of claim 1 , wherein the amplified DNA is tagged with up to 1024 molecular barcodes.
6 . The method of claim 1 , wherein the amplified DNA is tagged with between 1024 to 65536 molecular barcodes.
7 . The method of claim 1 , wherein the biological sample is not obtained from a pregnant woman.
8 . The method of claim 1 , wherein 10 to 200 target loci are pre-amplified in a single reaction volume.
9 . The method of claim 1 , further comprising tagging the amplified DNA with a sample-specific barcode.
10 . The method of claim 1 , wherein the amplified DNA is tagged by ligation.
11 . The method of claim 1 , further comprising identifying the SNV loci with a limit of detection of less than 0.01%.
12 . The method of claim 1 , wherein the method does not comprise PCR amplification in a microfluidic device.