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 measuring an amount of DNA from a first individual in a biological sample of a second individual, comprising:
pre-amplifying at least 50 polymorphic loci from cell-free DNA of mixed origin using in a single reaction volume to obtain pre-amplified DNA, wherein the cell-free DNA is extracted from the biological sample and comprises DNA from the first individual and DNA from the second individual;
dividing the pre-amplified DNA into multiple aliquots; amplifying subpools of the polymorphic loci in parallel in individual reaction volumes to obtain amplified DNA, wherein each reaction volume comprises at least one aliquot of the pre-amplified DNA; and pooling the amplified DNA into one pool;
performing a barcoding PCR to add a sequencing tag and a sample index to the amplified DNA;
sequencing the amplified DNA obtained from multiple samples together in a single sequencing lane, and for each sample measuring an amount of each allele at the polymorphic loci; and
determining the amount of the DNA from the first individual in the biological sample.
2 . The method of claim 1 , wherein the biological sample is a blood, serum, plasma, or urine sample.
3 . The method of claim 1 , wherein the cell-free DNA of mixed origin comprises DNA from a transplant.
4 . The method of claim 1 , wherein the cell-free DNA of mixed origin comprises DNA from a fetus.
5 . The method of claim 1 , wherein the pre-amplifying step comprises 10-30 PCR cycles.
6 . The method of claim 1 , wherein the pre-amplifying step comprises 15 PCR cycles.
7 . The method of claim 1 , wherein 50-5,000 polymorphic loci are pre-amplified in a single reaction volume.
8 . The method of claim 1 , wherein 50-500 polymorphic loci are pre-amplified in a single reaction volume.
9 . The method of claim 1 , wherein more than 100 polymorphic loci are pre-amplified in a single reaction volume.
10 . The method of claim 1 , wherein more than 200 polymorphic loci are pre-amplified in a single reaction volume.
11 . The method of claim 1 , wherein the polymorphic loci are SNP loci.
12 . The method of claim 1 , wherein the polymorphic loci comprise SNP loci on chromosome 1.
13 . The method of claim 1 , wherein the polymorphic loci comprise SNP loci on chromosome 2.
14 . The method of claim 1 , wherein the polymorphic loci comprise SNP loci on chromosome 3.
15 . The method of claim 1 , wherein the sequencing is high-throughput sequencing.
16 . The method of claim 1 , wherein the sequencing is sequencing-by-synthesis.
17 . The method of claim 1 , wherein the method is performed without prior knowledge of genotypes of the first and second individuals.