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 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.