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 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 are 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; 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.
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 at least one amplicon comprises two or more nearby single nucleotide polymorphism or variant loci.
4 . The method of claim 1 , wherein the plurality of target loci comprises between 100 and 2,000 single nucleotide polymorphism or variant loci.
5 . The method of claim 1 , wherein the plurality of target loci comprises between 200 and 1,000 single nucleotide polymorphism or variant loci.
6 . The method of claim 1 , wherein the plurality of target loci comprises between 300 and 2,000 single nucleotide polymorphism or variant loci.
7 . The method of claim 1 , wherein the cell-free DNA are tagged with up to 1024 molecular barcodes.
8 . The method of claim 1 , wherein the cell-free DNA are tagged with 1024-65536 molecular barcodes.
9 . The method of claim 1 , wherein the cell-free DNA are tagged with the molecular barcodes through adaptor ligation.
10 . The method of claim 1 , wherein sequence reads originating from the same original molecule are identified using the molecular barcodes.
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 a library of DNA, wherein the cell-free DNA from a single biological sample are 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; 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.
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 at least one amplicon comprises two or more nearby single nucleotide polymorphism or variant loci.
14 . The method of claim 11 , wherein the plurality of target loci comprises between 100 and 2,000 single nucleotide polymorphism or variant loci.
15 . The method of claim 11 , wherein the plurality of target loci comprises between 200 and 1,000 single nucleotide polymorphism or variant loci.
16 . The method of claim 11 , wherein the plurality of target loci comprises between 300 and 2,000 single nucleotide polymorphism or variant loci.
17 . The method of claim 11 , wherein the cell-free DNA are tagged with up to 1024 molecular barcodes.
18 . The method of claim 11 , wherein the cell-free DNA are tagged with 1024-65536 molecular barcodes.
19 . The method of claim 11 , wherein the cell-free DNA are tagged with the molecular barcodes through adaptor ligation.
20 . The method of claim 11 , wherein sequence reads originating from the same original molecule are identified using the molecular barcodes.