METHODS FOR TARGETED SEQUENCING OF CELL-FREE DNA
The invention provides methods for simultaneously enriching multiple target regions of interest in one reaction volume, from cell-free DNA isolated from a blood or plasma sample, followed by high-thought sequencing and sequence read analysis. The invention also provides library of target-specific oligonucletotide primers or probes for the multiplexed target enrichment.
1 . A method for targeted sequencing of cell-free DNA, comprising:
appending at least one adaptor to cell-free DNA extracted from a blood or plasma sample of a human subject to generate adapted DNA;
performing targeted enrichment on the adapted DNA, wherein the targeted enrichment comprises (i) contacting adapted DNA with a library comprising oligonucleotides that specifically hybridize to at least 800 different target loci, wherein a target locus comprises a variant position, and (ii) amplifying adapted DNA that was contacted in step (i) in the same reaction volume to generate enriched DNA; and
determining the sequence of at least some of the enriched DNA or DNA derived therefrom by performing high-throughput sequencing to generate sequence reads.
2 . The method of claim 1 , wherein the method further comprises analyzing the sequence reads and identifying one or more variants from the sequence reads.
3 . The method of claim 1 , wherein the cell-free DNA comprises cell-free DNA derived from both normal and cancer cells of the human subject.
4 . The method of claim 1 , wherein the library comprises oligonucleotides that specifically hybridize to at least 1,200 different target loci comprising single nucleotide variant positions on one or more chromosomes.
5 . The method of claim 1 , wherein the library comprises oligonucleotides that specifically hybridize to 1,000 to 20,000 different target loci comprising single nucleotide variant positions on one or more chromosomes.
6 . The method of claim 1 , wherein the library comprises oligonucleotides that specifically hybridize to 1,000 to 10,000 different target loci comprising single nucleotide variant positions on one or more chromosomes.
7 . The method of claim 1 , wherein the concentration of each target locus-specific oligonucleotide in the library is 5 nM or less.
8 . The method of claim 1 , wherein the high-throughput sequencing is sequencing-by-synthesis.
9 . The method of claim 1 , wherein at least 90% of the sequence reads comprise the variants at loci specifically hybridized by the oligonucleotides.
10 . The method of claim 1 , wherein at least 95% of the sequence reads comprise the variants at loci specifically hybridized by the oligonucleotides.
11 . The method of claim 1 , wherein the at least one adaptor comprises a universal priming sequence.
12 . The method of claim 11 , wherein the method further comprises amplifying adapted DNA using the universal priming sequence prior to performing targeted enrichment.
13 . A method for targeted sequencing of cell-free DNA, comprising:
tagging cell-free DNA extracted from a biological sample from a human subject with molecular barcodes to generate tagged DNA;
performing targeted enrichment on the tagged DNA, wherein the targeted enrichment comprises (i) contacting tagged DNA with a library comprising oligonucleotides that specifically hybridize to at least 800 different target loci, wherein a target locus comprises a variant position, and (ii) amplifying tagged DNA that was contacted in step (i) in the same reaction volume to generate enriched DNA;
determining the sequence of at least some of the enriched DNA or DNA derived therefrom by performing high-throughput sequencing to generate sequence reads.
14 . The method of claim 13 , wherein the method further comprises analyzing the sequence reads and identifying one or more variants from the sequence reads.
15 . The method of claim 13 , wherein the cell-free DNA comprises cell-free DNA derived from both normal and cancer cells of the human subject.
16 . The method of claim 13 , wherein the library comprises oligonucleotides that specifically hybridize to at least 1,200 different target loci comprising single-nucleotide-variant positions on one or more chromosomes.
17 . The method of claim 13 , wherein the library comprises oligonucleotides that specifically hybridize to 1,000 to 20,000 different target loci comprising single-nucleotide-variant positions on one or more chromosomes.
18 . The method of claim 13 , wherein the library comprises oligonucleotides that specifically hybridize to 1,000 to 10,000 different target loci comprising single-nucleotide-variant positions on one or more chromosomes.
19 . The method of claim 13 , wherein the concentration of each target-specific oligonucleotide in the library of target-specific oligonucleotides is 5 nM or less.
20 . The method of claim 13 , wherein the high-throughput sequencing is sequencing-by-synthesis.
21 . The method of claim 13 , wherein at least 90% of the sequence reads comprises the variants at loci specifically hybridized by the oligonucleotides.
22 . The method of claim 13 , wherein at least 95% of the sequence reads comprises the variants at loci specifically hybridized by the oligonucleotides.
23 . The method of claim 13 , wherein the tagged DNA are tagged with up to 1024 different molecular barcodes.
24 . The method of claim 13 , wherein the tagged DNA are tagged with 1024 to 65536 different molecular barcodes.