DETECTING MUTATIONS AND PLOIDY IN CHROMOSOMAL SEGMENTS
The invention provides methods, systems, and computer readable medium for detecting ploidy of chromosome segments or entire chromosomes, for detecting single nucleotide variants and for detecting both ploidy of chromosome segments and single nucleotide variants. In some aspects, the invention provides methods, systems, and computer readable medium for detecting cancer or a chromosomal abnormality in a gestating fetus.
1 . A method for preparing a sample of a subject having cancer or suspected of having cancer useful for identifying one or more tumor-specific variants in a biological sample of the subject, the method comprising:
(a) selectively enriching 100 to 100,000 target loci from a first cell-free DNA sample obtained from a first biological sample of the subject to obtain a first set of selectively enriched DNA molecules, wherein the 100 to 100,000 target loci span 100 to 100,000 tumor-specific variants previously identified from a tumor biopsy sample of the subject; and
(b) determining the sequence of at least some of the first set of selectively enriched DNA molecules and obtaining sequence reads with a depth of read of 20,000 to 500,000 per target locus for at least 100 of the target loci, and identifying one or more of the tumor-specific variants present in the first cell-free DNA sample from the sequence reads, wherein the tumor-specific variants comprise one or more duplications, deletions, inversions, translocations, or a combination thereof.
2 . The method of claim 1 , wherein the tumor biopsy sample of the subject includes a tumor tissue from a solid tumor.
3 . The method of claim 1 , wherein the first cell-free DNA sample is obtained from a blood, plasma, serum, or urine sample of the subject.
4 . The method of claim 1 , wherein the first cell-free DNA sample comprises circulating tumor DNA.
5 . The method of claim 1 , wherein step (a) comprises selectively enriching 100 to 1,000 target loci, wherein the 100 to 1,000 target loci span 100 to 1,000 tumor-specific variants previously identified from a tumor biopsy sample of the subject, wherein the selective enrichment of the target loci is performed using target locus-specific primers or probes in one reaction volume, wherein the first set of selectively enriched DNA molecules are tagged with a plurality of different molecular barcodes.
6 . The method of claim 1 , wherein step (a) comprises selectively enriching 100 to 200 target loci, wherein the 100 to 200 target loci span 100 to 200 tumor-specific variants previously identified from a tumor biopsy sample of the subject, wherein the selective enrichment of the target loci is performed using target locus-specific primers or probes in one reaction volume, wherein the first set of selectively enriched DNA molecules are tagged with a plurality of different molecular barcodes.
7 . The method of claim 1 , wherein step (b) comprises identifying at least one duplication or deletion present in the first cell-free DNA sample from the sequence reads.
8 . The method of claim 1 , wherein step (b) comprises identifying at least one inversion or translocation present in the first cell-free DNA sample from the sequence reads.
9 . The method of claim 1 , wherein step (b) further comprises identifying at least one single nucleotide variant present in the first cell-free DNA sample from the sequence reads.
10 . The method of claim 1 , wherein step (b) comprises determining the sequence of at least some of the first set of selectively enriched DNA molecules and obtaining sequence reads with a depth of read of 20,000 to 250,000 per target locus for at least 100 of the target loci.
11 . The method of claim 1 , wherein the subject is a human subject.
12 . The method of claim 1 , wherein the cancer is colorectal cancer, lung cancer, bladder cancer, or breast cancer.
13 . The method of claim 1 , wherein the method further comprises performing barcoding PCR prior to step (b).
14 . The method of claim 1 , wherein the method further comprises the steps of:
selectively enriching 100 to 100,000 target loci from a second cell-free DNA sample obtained from a second biological sample of the subject to obtain a second set of selectively enriched DNA molecules, wherein the 100 to 100,000 target loci span at least one of the 100 to 100,000 tumor-specific variants previously identified from a tumor biopsy sample of the subject; and
determining the sequence of at least some of the second set of selectively enriched DNA molecules and obtaining sequence reads with a depth of read of 20,000 to 500,000 per target locus for at least 100 of the target loci, and identifying one or more of the tumor-specific variants present in the second cell-free DNA sample from the sequence reads.
15 . The method of claim 14 , wherein the first biological sample and the second biological sample are taken from the same subject at different points in time.
16 . The method of claim 14 , wherein the first biological sample and the second biological sample are taken from the same subject from different sources.
17 . The method of claim 14 , wherein the method further comprises detecting recurrence and/or metastases of the cancer from the tumor-specific variants detected in the first cell-free DNA sample or the second cell-free DNA sample.
18 . The method of claim 14 , wherein the method identifies a tumor-specific variant present in the first cell-free DNA sample or the second cell-free DNA sample at a limit of detection of less than or equal to 0.015%.
19 . The method of claim 18 , wherein the limit of detection is calculated by LOD-mr5, LOD-zs5.0, or LOD-zs5.0-mr5.
20 . A method for preparing a sample of a subject having cancer or suspected of having cancer useful for identifying one or more tumor-specific variants in a biological sample of the subject, the method comprising:
(a) selectively enriching 100 to 100,000 target loci from a first cell-free DNA sample obtained from a biological sample of the subject to obtain a first set of selectively enriched DNA molecules, wherein the 100 to 100,000 target loci span 100 to 100,000 tumor-specific variants previously identified from a tumor biopsy sample of the subject; and
(b) determining the sequence of at least some of the first set of selectively enriched DNA molecules and obtaining sequence reads, and identifying one or more of the tumor-specific variants present in the first cell-free DNA sample from the sequence reads, wherein the method identifies a tumor-specific variant present in the first cell-free DNA sample at a limit of detection of less than or equal to 0.015%, wherein the tumor-specific variants comprise one or more duplications, deletions, inversions, translocations, or a combination thereof.
21 . The method of claim 20 , wherein the tumor biopsy sample of the subject includes a tumor tissue from a solid tumor, wherein the first cell-free DNA sample is obtained from a blood, plasma, serum, or urine sample of the subject.
22 . The method of claim 20 , wherein step (b) comprises identifying at least one duplication or deletion present in the first cell-free DNA sample from the sequence reads.
23 . The method of claim 20 , wherein step (b) comprises identifying at least one inversion or translocation present in the first cell-free DNA sample from the sequence reads.
24 . The method of claim 20 , wherein step (b) further comprises identifying at least one single nucleotide polymorphism present in the first cell-free DNA sample from the sequence reads.
25 . The method of claim 20 , wherein step (b) comprises determining the sequence of at least some of the first set of selectively enriched DNA molecules and obtaining sequence reads with a depth of read of 20,000 to 250,000 per target locus.
26 . The method of claim 1 , further comprising identifying the tumor-specific variants from the tumor biopsy sample by whole genome sequencing.
27 . The method of claim 1 , further comprising identifying the tumor-specific variants from the tumor biopsy sample by whole exome sequencing.
28 . The method of claim 1 , wherein step (b) comprises identifying at least 10 tumor-specific variants from the sequence reads.
29 . The method of claim 1 , wherein step (b) comprises identifying from the sequence reads at least two of the tumor-specific variants that are phased alleles and less than 0.01 kb from each other.
30 . The method of claim 1 , wherein the tumor-specific variants comprise clonal tumor-specific variants identified by sequencing nucleic acids from multiple regions of the tumor biopsy sample.