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.
Claims20
A method for enriching and sequencing cell-free DNA, comprising:
- tagging cell-free DNA isolated from a biological sample with molecular barcodes to obtain a library of DNA, wherein the cell-free DNA from a single biological sample is tagged with a plurality of molecular barcodes;
- amplifying the library of DNA and enriching for a plurality of target loci using a plurality of target-specific hybrid capture probes to obtain an enriched library of amplicons, wherein at least one amplicon comprises two or more target loci, wherein the two or more target loci comprise two or more single nucleotide polymorphism or variant loci within 50 bp of each other;
- and
- performing high-throughput sequencing to sequence the amplicons to obtain sequence reads of at least 50 target loci and determining whether the at least 50 target loci comprise a cancer-associated mutation based on the sequence reads, and wherein determining whether the at least 50 target loci comprise a cancer-associated mutation is based at least in part on the two or more single nucleotide polymorphism or variant loci within 50 bp of each other.
A method for enriching and sequencing cell-free DNA, comprising:
- tagging cell-free DNA isolated from a biological sample with molecular barcodes to obtain a library of DNA, wherein the cell-free DNA from a single biological sample is tagged with a plurality of molecular barcodes;
- enriching the library of DNA for a plurality of target loci using a plurality of target-specific hybrid capture probes and amplifying the target loci to obtain an enriched library of amplicons, wherein at least one amplicon comprises two or more target loci, wherein the two or more target loci comprise two or more single nucleotide polymorphism or variant loci within 50 bp of each other;
- and
- performing high-throughput sequencing to sequence the amplicons to obtain sequence reads of at least 50 target loci and determine whether the target loci comprise a cancer-associated mutation based on the sequence reads, and wherein determining whether the at least 50 target loci comprise a cancer-associated mutation is based at least in part on the two or more single nucleotide polymorphism or variant loci within 50 bp of each other.
Dependencies are inferred from the claim text.