Methods for simultaneous amplification of target loci
AssigneeNatera, Inc.
InventorsMatthew Rabinowitz, Matthew Micah Hill, Bernhard Zimmermann, Johan Baner, George Gemelos, Milena Banjevic, Allison Ryan, Styrmir Sigurjonsson, Zachary Demko
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Abstract
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.
Claims21
A method for preparing a non-naturally occurring composition from a biological sample useful for measuring an amount of DNA from a first individual in the biological sample of a second individual, comprising:
- pre-amplifying at least 50 polymorphic loci from cell-free DNA in a single reaction volume to obtain a non-naturally occurring composition of pre-amplified DNA of mixed origin, wherein the cell-free DNA is extracted from the biological sample and comprises DNA from the first individual and DNA from the second individual, wherein the DNA from the first individual comprises DNA from a transplant;
- performing targeted multiplex PCR amplification of the pre-amplified DNA at the at least 50 polymorphic loci using at least 50 PCR primers to obtain amplified DNA;
- and
- barcoding the amplified DNA to obtain a barcoded sequencing library of the amplified DNA to prepare the amplified DNA for high-throughput sequencing,
- wherein the method is performed without prior knowledge of genotypes of the first and second individuals.
A method for preparing a non-naturally occurring composition from a biological sample useful for measuring an amount of DNA from a first individual in the biological sample of a second individual, comprising:
- pre-amplifying at least 50 SNP loci from cell-free DNA in a single reaction volume to obtain a non-naturally occurring composition of pre-amplified DNA of mixed origin, wherein the cell-free DNA is extracted from the biological sample and comprises DNA from the first individual and DNA from the second individual, wherein the DNA from the first individual comprises DNA from a transplant;
- performing targeted multiplex PCR amplification on the pre-amplified DNA at the at least 50 SNP loci using at least 50 PCR primers to obtain amplified DNA;
- performing a barcoding PCR on the amplified DNA to obtain a barcoded sequencing library such that multiple samples can be sequenced together in a single sequencing lane by high-throughput sequencing,
- wherein the method is performed without prior knowledge of genotypes of the first and second individuals.
Dependencies are inferred from the claim text.