METHODS FOR PREPARING DNA REFERENCE MATERIALS AND CONTROLS
Disclosed are controls and reference materials for use in identifying any of a number of genotypes and/or for use in identifying or characterizing a disease or condition. The controls and reference materials may be particularly useful for diagnostic tests that utilize circulating cell-free DNA (cfDNA). Also disclosed herein are methods of generating large quantities of cfDNA that preserve the original cfDNA size and input sequences. In some embodiments, the methods comprise amplifying, digesting, and purifying cfDNA isolated from a subject. The cfDNA may be circulating tumor DNA (“ctDNA). The methods disclosed herein result in a cfDNA or ctDNA clone library that is significantly more “commutable” compared to existing methods.
1 . A control for use in identifying a genotype, comprising amplified cell-free DNA (cfDNA), and a first mixture of nucleic acids; wherein the cfDNA has a base pair (bp) length of about 75 bps to about 600 bps, the first mixture of nucleic acids comprises a nucleotide sequence that encodes the genotype, and the first mixture of nucleic acids constitutes at least 50% of double stranded DNA of about 75 bps to about 600 bps in length.
2 . The control of claim 1 , wherein the first mixture of nucleic acids encodes substantially all of a human genome.
3 . The control of claim 1 , further comprising a second mixture of nucleic acids comprising a nucleotide sequence that encodes a second genotype, wherein the genotype and the second genotype are alternate genotypes that occur at the same genetic locus.
4 . The control of claim 3 , wherein the second mixture of nucleic acids encodes substantially all of a second human genome.
5 . The control of claim 3 , wherein the ratio of the copy number of the nucleotide sequence that encodes the genotype to the copy number of the nucleotide sequence that encodes the second genotype is about 1:1000 to about 1000:1.
6 . The control of claim 1 , wherein the genotype is associated with a neoplasm.
7 . The control of claim 1 , wherein the genotype is associated with a provirus.
8 . The control of claim 1 , wherein the genotype is associated with a hereditary disease.
9 . The control of claim 1 , wherein the genotype is associated with a somatic mutation.
10 . The control of claim 9 , wherein the genotype is a mutation to a gene selected from the group consisting of MTOR, MPL, NRAS, PARP1, AKT3, DNMT3A, MSH2, IDH1, VHL, MLH1, MYD88, CTNNB1, ATR, PIK3CA, FGFR3, PDGFRA, KIT, FBXW7, APC, GABRG2, NPM1, EGFR, MET, BRAF, EZH2, JAK2, GNAQ, RET, PTEN, ATM, KRAS, PTPN11, FLT3, RB1, PARP2, ARHGAP5, AKT1, RAD51, IDH2, TP53, NF1, SMAD4, AKT2, ERCC1, and GNAS.
11 . The control of claim 9 , wherein the genotype is a mutation to a gene selected from the group consisting of AKT1, ATM, BRAF, CDKN2A, CSF1R, EGFR, ERBB2, ERBB4, FGFR1, FGFR2, FGFR3, GNA11, HRAS, JAK2, JAK3, KDR, KIT, KRAS, MET, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, and STK11.
12 . The control of claim 1 , wherein the genotype is associated with a virus or bacteria.
13 . The control of claim 1 , wherein the genotype is associated with a graft rejection.
14 . The control of claim 1 , wherein the second genotype is not associated with a disease.
15 . The control of claim 1 , wherein the genotype is a single nucleotide polymorphism, point mutation, premature stop codon, trinucleotide repeat, translocation, somatic rearrangement, allelomorph, single nucleotide variant, coding insertion or deletion (“indel”), splice variant, regulatory variant, copy number variant, or gene fusion.
16 - 47 . (canceled)
48 . A control for use in determining the ploidy of a chromosome in a fetus, comprising:
a first mixture of nucleic acids comprising a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence has sequence homology with the chromosome; the second nucleotide sequence has sequence homology with a different chromosome; and the ratio of the copy number of the first nucleotide sequence to the copy number of the second nucleotide sequence is greater than 1:1;
a second mixture of nucleic acids comprising the first nucleotide sequence and the second nucleotide sequence, wherein the ratio of the copy number of the first nucleotide sequence to the copy number of the second nucleotide sequence is about 1:1; and
a sample of cfDNA, wherein the cfDNA has a base pair (bp) length of about 75 bps to about 600 bps,
wherein the first mixture of nucleic acids constitutes at least 50% of double stranded DNA of about 75 bps to about 600 bps in length.
49 - 109 . (canceled)
110 . A method for quantitative analysis of cell-free DNA (cfDNA), comprising:
(a) treating the 5′ end or 3′ end or both of cfDNA with one or more end-repair enzymes to generate end-repaired cfDNA;
(b) ligating one or more adaptors, wherein the adaptors comprise a restriction enzyme site and a sequence from a respiratory syncytial virus (RSV), to each end of the end-repaired cfDNA to generate a cfDNA library;
(c) amplifying the cfDNA library to generate a cfDNA library clone;
(d) digesting the cfDNA library clone with a restriction enzyme to generate a modified cfDNA clone library; wherein digesting the cfDNA library clones with the restriction enzyme removes the one or more adaptors; and
(e) purifying the modified cfDNA clone library.
111 - 170 . (canceled)
171 . A method of predicting, diagnosing, or monitoring a genetic disease in a subject comprising:
(a) isolating or obtaining a sample of cfDNA from a subject;
(b) treating the 5′ end or 3′ end or both of cfDNA with one or more end-repair enzymes to generate end-repaired cfDNA;
(c) ligating one or more adaptors, wherein the adaptors comprise a restriction enzyme site and a sequence from a respiratory syncytial virus (RSV), to each end of the end-repaired cfDNA to generate a cfDNA library;
(d) amplifying the cfDNA library to generate a cfDNA library clone;
(e) digesting the cfDNA library clone with a restriction enzyme to generate a modified cfDNA clone library; wherein the digesting the cfDNA library clone with the restriction enzyme removes the one or more adaptors; and
(f) purifying the modified cfDNA clone library.
172 - 220 . (canceled)