Methods for simultaneous amplification of target loci

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.

What is claimed is: 
     
         1 . A method for enriching and sequencing cell-free DNA, comprising:
 tagging each strand of cell-free DNA isolated from a biological sample with a molecular barcode to obtain barcoded DNA;   amplifying the barcoded DNA by universal amplification to obtain a sequencing library;   enriching for a plurality of target loci from the sequencing library using hybrid capture probes; and   performing high-throughput sequencing to sequence the enriched target loci and determine whether the target loci comprise a cancer-associated mutation.   
     
     
         2 . The method of  claim 1 , wherein the biological sample is a blood, plasma, serum, or urine sample. 
     
     
         3 . The method of  claim 1 , wherein the plurality of target loci comprises between 100 and 2,000 SNV loci. 
     
     
         4 . The method of  claim 1 , wherein the plurality of target loci comprises between 200 and 1,000 SNV loci. 
     
     
         5 . The method of  claim 1 , wherein the plurality of target loci comprises between 300 and 2,000 SNV loci. 
     
     
         6 . The method of  claim 1 , wherein the cell-free DNA are tagged with up to 1024 molecular barcodes. 
     
     
         7 . The method of  claim 1 , wherein the cell-free DNA are tagged with 1024-65536 molecular barcodes. 
     
     
         8 . The method of  claim 1 , wherein the cell-free DNA are tagged with the molecular barcodes through ligation. 
     
     
         9 . The method of  claim 1 , wherein sequence reads originating from the same original molecule are identified using the molecular barcodes. 
     
     
         10 . The method of  claim 1 , wherein the universal amplification introduces a sample-specific barcode, and wherein amplified DNAs of multiple samples are pooled together and sequenced in a single sequencing lane. 
     
     
         11 . A method for enriching and sequencing cell-free DNA, comprising:
 tagging cell-free DNA isolated from a biological sample with molecular barcodes to obtain barcoded DNA;   amplifying the barcoded DNA by universal amplification to obtain a sequencing library;   enriching for a plurality of target loci from the sequencing library using hybrid capture probes; and   performing high-throughput sequencing to sequence the enriched target loci and determine whether the target loci comprise a cancer-associated mutation.   
     
     
         12 . The method of  claim 11 , wherein the biological sample is a blood, plasma, serum, or urine sample. 
     
     
         13 . The method of  claim 11 , wherein the plurality of target loci comprises between 100 and 2,000 polymorphic loci. 
     
     
         14 . The method of  claim 11 , wherein the plurality of target loci comprises between 200 and 1,000 polymorphic loci. 
     
     
         15 . The method of  claim 11 , wherein the plurality of target loci comprises between 300 and 2,000 polymorphic loci. 
     
     
         16 . The method of  claim 11 , wherein the cell-free DNA are tagged with up to 1024 molecular barcodes. 
     
     
         17 . The method of  claim 11 , wherein the cell-free DNA are tagged with 1024-65536 molecular barcodes. 
     
     
         18 . The method of  claim 11 , wherein the cell-free DNA are tagged with the molecular barcodes through ligation. 
     
     
         19 . The method of  claim 11 , wherein sequence reads originating from the same original molecule are identified using the molecular barcodes. 
     
     
         20 . The method of  claim 11 , wherein the universal amplification introduces a sample-specific barcode, and wherein amplified DNAs of multiple samples are pooled together and sequenced in a single sequencing lane.