METHODS FOR DETERMINING COPY NUMBER VARIATIONS
The invention provides a method for determining copy number variations (CNV) of a sequence of interest in a test sample that comprises a mixture of nucleic acids that are known or are suspected to differ in the amount of one or more sequence of interest. The method comprises a statistical approach that accounts for accrued variability stemming from process-related, interchromosomal and inter-sequencing variability. The method is applicable to determining CNV of any fetal aneuploidy, and CNVs known or suspected to be associated with a variety of medical conditions.
1 . A method for identifying fetal trisomy 21, said method comprising the steps:
obtaining sequence information for a plurality of fetal and maternal nucleic acid molecules of a maternal blood sample;
using the sequence information to identify a number of mapped sequence tags for chromosome 21;
using the sequence information to identify a number of mapped sequence tags for at least one normalizing chromosome;
using the number of mapped sequence tags identified for chromosome 21 in step (b) and the number of mapped sequence tags identified for the at least one normalizing chromosome in step (c) to calculate a chromosome dose for chromosome 21; and
comparing said chromosome dose to at least one threshold value, and thereby identifying the presence or absence of fetal trisomy 21.
2 . The method of claim 1 , further comprising sequencing at least a portion of said nucleic acid molecules to obtain sequence information for a plurality of fetal and maternal nucleic acid molecules of a maternal blood sample.
3 . The method of claim 1 or claim 2 , wherein step (d) comprises calculating a chromosome dose for chromosome 21 as the ratio of the number of mapped sequence tags identified for chromosome 21 and the number of mapped sequence tags identified for the at least one normalizing chromosome.
4 . The method of claim 1 or claim 2 , wherein step (d) comprises:
calculating a sequence tag density ratio for chromosome 21, by relating the number of mapped sequence tags identified for chromosome 21 in step (b) to the length of chromosome 21;
calculating a sequence tag density ratio for said at least one normalizing chromosome, by relating the number of mapped sequence tags identified for said at least one normalizing chromosome in step (c) to the length of said at least one normalizing chromosome; and
using the sequence tag density ratios calculated in steps (i) and (ii) to calculate a chromosome dose for chromosome 21, wherein the chromosome dose is calculated as the ratio of the sequence tag density ratio for chromosome 21 and the sequence tag density ratio for said at least one normalizing chromosome.
5 . The method of claim 1 or claim 2 , wherein said at least one normalizing chromosome is a chromosome having the smallest variability and/or the greatest differentiability.
6 . The method of claim 1 or claim 2 , wherein said at least one normalizing chromosome is selected from chromosome 9, chromosome 1, chromosome 2, chromosome 11, chromosome 12, and chromosome 14.
7 . The method of claim 1 or claim 2 , wherein said at least one normalizing chromosome is a group of chromosomes selected from chromosome 9, chromosome 1, chromosome 2, chromosome 11, chromosome 12, and chromosome 14.
8 . The method of claim 1 or claim 2 , wherein said fetal and maternal nucleic acid molecules are cell-free DNA molecules.
9 . The method of claim 1 or claim 2 , wherein said sequencing is next generation sequencing (NGS).
10 . The method of claim 1 or claim 2 , wherein said sequencing is massively parallel sequencing using sequencing-by-synthesis with reversible dye terminators.
11 . The method of claim 1 or claim 2 , wherein said sequencing is sequencing-by-ligation.
12 . The method of claim 1 or claim 2 , wherein said sequencing comprises an amplification.
13 . The method of claim 1 or claim 2 , wherein said sequencing is single molecule sequencing.
14 . A method for identifying fetal trisomy 18, said method comprising the steps:
obtaining sequence information for a plurality of fetal and maternal nucleic acid molecules of a maternal blood sample;
using the sequence information to identify a number of mapped sequence tags for chromosome 18;
using the sequence information to identify a number of mapped sequence tags for at least one normalizing chromosome;
using the number of mapped sequence tags identified for chromosome 18 in step (b) and the number of mapped sequence tags identified for the at least one normalizing chromosome in step (c) to calculate a chromosome dose for chromosome 18; and
comparing said chromosome dose to at least one threshold value, and thereby identifying the presence or absence of fetal trisomy 18;
15 . The method of claim 14 , further comprising sequencing at least a portion of said nucleic acid molecules, to obtain sequence information for a plurality of fetal and maternal nucleic acid molecules of a maternal blood sample.
16 . The method of claim 14 or claim 15 , wherein step (d) comprises calculating a chromosome dose for chromosome 18 as the ratio of the number of mapped sequence tags identified for chromosome 18 and the number of mapped sequence tags identified for the at least one normalizing chromosome.
17 . The method of claim 14 or claim 15 , wherein step (d) comprises:
calculating a sequence tag density ratio for chromosome 18, by relating the number of mapped sequence tags identified for chromosome 18 in step (b) to the length of chromosome 18;
calculating a sequence tag density ratio for said at least one normalizing chromosome, by relating the number of mapped sequence tags identified for said at least one normalizing chromosome in step (c) to the length of said at least one normalizing chromosome; and
using the sequence tag density ratios calculated in steps (i) and (ii) to calculate a chromosome dose for chromosome 18, wherein the chromosome dose is calculated as the ratio of the sequence tag density ratio for chromosome 18 and the sequence tag density ratio for said at least one normalizing chromosome.
18 . The method of claim 14 or claim 15 , wherein said at least one normalizing chromosome is a chromosome having the smallest variability and/or the greatest differentiability.
19 . The method of claim 14 or claim 15 , wherein said at least one normalizing chromosome is selected from chromosome 8, chromosome 2, chromosome 3, chromosome 5, chromosome 6, chromosome 12, and chromosome 14.
20 . The method of claim 14 or claim 15 , wherein said at least one normalizing chromosome is a group of chromosomes selected from chromosome 8, chromosome 2, chromosome 3, chromosome 5, chromosome 6, chromosome 12, and chromosome 14.
21 . The method of claim 14 or claim 15 , wherein said fetal and maternal nucleic acid molecules are cell-free DNA molecules.
22 . The method of claim 14 or claim 15 , wherein said sequencing is next generation sequencing (NGS);
23 . The method of claim 14 or claim 15 , wherein said sequencing is massively parallel sequencing using sequencing-by-synthesis with reversible dye terminators.
24 . The method of claim 14 or claim 15 , wherein said sequencing is sequencing-by-ligation.
25 . The method of claim 14 or claim 15 , wherein said sequencing comprises an amplification.
26 . The method of claim 14 or claim 15 , wherein said sequencing is single molecule sequencing.
27 . A method for identifying fetal trisomy 13, said method comprising the steps:
obtaining sequence information for a plurality of fetal and maternal nucleic acid molecules of a maternal blood sample;
using the sequence information to identify a number of mapped sequence tags for chromosome 13;
using the sequence information to identify a number of mapped sequence tags for at least one normalizing chromosome;
using the number of mapped sequence tags identified for chromosome 13 in step (b) and the number of mapped sequence tags identified for the at least one normalizing chromosome in step (c) to calculate a chromosome dose for chromosome 13; and
comparing said chromosome dose to at least one threshold value, and thereby identifying the presence or absence of fetal trisomy 13;
28 . The method of claim 27 , further comprising sequencing at least a portion of said nucleic acid molecules, to obtain sequence information for a plurality of fetal and maternal nucleic acid molecules of a maternal blood sample.
29 . The method of claim 27 or claim 28 , wherein step (d) comprises calculating a chromosome dose for chromosome 13 as the ratio of the number of mapped sequence tags identified for chromosome 13 and the number of mapped sequence tags identified for the at least one normalizing chromosome.
30 . The method of claim 27 or claim 28 , wherein step (d) comprises:
calculating a sequence tag density ratio for chromosome 13, by relating the number of mapped sequence tags identified for chromosome 13 in step (b) to the length of chromosome 13;
calculating a sequence tag density ratio for said at least one normalizing chromosome, by relating the number of mapped sequence tags identified for said at least one normalizing chromosome in step (c) to the length of said at least one normalizing chromosome; and
using the sequence tag density ratios calculated in steps (i) and (ii) to calculate a chromosome dose for chromosome 13, wherein the chromosome dose is calculated as the ratio of the sequence tag density ratio for chromosome 13 and the sequence tag density ratio for said at least one normalizing chromosome.
31 . The method of claim 27 or claim 28 , wherein said at least one normalizing chromosome is a chromosome having the smallest variability and/or the greatest differentiability.
32 . The method of claim 27 or claim 28 , wherein said at least one normalizing chromosome is selected from chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8.
33 . The method of claim 27 or claim 28 , wherein said at least one normalizing chromosome is a group of chromosomes selected from chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8.
34 . The method of claim 27 or claim 28 , wherein said fetal and maternal nucleic acid molecules are cell-free DNA molecules.
35 . The method of claim 27 or claim 28 , wherein said sequencing is (i) next generation sequencing (NGS).
36 . The method of claim 27 or claim 28 , wherein said sequencing is massively parallel sequencing using sequencing-by-synthesis with reversible dye terminators.
37 . The method of claim 27 or claim 28 , wherein said sequencing is sequencing-by-ligation.
38 . The method of claim 27 or claim 28 , wherein said sequencing comprises an amplification.
39 . The method of claim 27 or claim 28 , wherein said sequencing is single molecule sequencing.
40 . A method for identifying fetal monosomy X, said method comprising the steps:
obtaining sequence information for a plurality of fetal and maternal nucleic acid molecules of a maternal blood sample;
using the sequence information to identify a number of mapped sequence tags for chromosome X;
using the sequence information to identify a number of mapped sequence tags for at least one normalizing chromosome;
using the number of mapped sequence tags identified for chromosome X in step (b) and the number of mapped sequence tags identified for the at least one normalizing chromosome in step (c) to calculate a chromosome dose for chromosome X; and
comparing said chromosome dose to at least one threshold value, and thereby identifying the presence or absence of fetal monosomy X.
41 . The method of claim 40 , further comprising sequencing at least a portion of said nucleic acid molecules, to obtain sequence information for a plurality of fetal and maternal nucleic acid molecules of a maternal blood sample.
42 . The method of claim 40 or claim 41 , wherein step (d) comprises calculating a chromosome dose for chromosome X as the ratio of the number of mapped sequence tags identified for chromosome X and the number of mapped sequence tags identified for the at least one normalizing chromosome.
43 . The method of claim 40 or claim 41 , wherein step (d) comprises:
calculating a sequence tag density ratio for chromosome X, by relating the number of mapped sequence tags identified for chromosome X in step (b) to the length of chromosome X;
calculating a sequence tag density ratio for said at least one normalizing chromosome, by relating the number of mapped sequence tags identified for said at least one normalizing chromosome in step (c) to the length of said at least one normalizing chromosome; and
using the sequence tag density ratios calculated in steps (i) and (ii) to calculate a chromosome dose for chromosome X, wherein the chromosome dose is calculated as the ratio of the sequence tag density ratio for chromosome X and the sequence tag density ratio for said at least one normalizing chromosome.
44 . The method of claim 40 or claim 41 , wherein said at least one normalizing chromosome is a chromosome having the smallest variability and/or the greatest differentiability.
45 . The method of claim 40 or claim 41 , wherein said at least one normalizing chromosome is selected from chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8.
46 . The method of claim 40 or claim 41 , wherein said at least one normalizing chromosome is a group of chromosomes selected from chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8.
47 . The method of claim 40 or claim 41 , wherein said fetal and maternal nucleic acid molecules are cell-free DNA molecules.
48 . The method of claim 40 or claim 41 , wherein said sequencing is next generation sequencing (NGS).
49 . The method of claim 40 or claim 41 , wherein said sequencing is massively parallel sequencing using sequencing-by-synthesis with reversible dye terminators.
50 . The method of claim 40 or claim 41 , wherein said sequencing is sequencing-by-ligation.
51 . The method of claim 40 or claim 41 , wherein said sequencing comprises an amplification.
52 . The method of claim 40 or claim 41 , wherein said sequencing is single molecule sequencing.
53 . A method for identifying fetal chromosomal aneuploidy in a test sample, said method comprising:
obtaining a test sample and a plurality of qualified samples, said test sample comprising a test nucleic acid molecules and said plurality of qualified samples comprising qualified nucleic acid molecules;
sequencing at least a portion of said qualified and test nucleic acid molecules, wherein said sequencing comprises providing a plurality of mapped sequence tags for a test and a qualified chromosome sequence of interest, and for at least one test and at least one qualified normalizing chromosome;
based on said sequencing of said qualified chromosome, calculating a qualified chromosome dose for said qualified chromosome of interest in each of said plurality of qualified samples, wherein said calculating a qualified chromosome dose comprises determining a parameter for said qualified chromosome of interest and at least one qualified normalizing chromosome;
based on said qualified chromosome dose, identifying at least one qualified normalizing chromosome, wherein said at least one qualified normalizing chromosome has the smallest variability and/or the greatest differentiability in sequence chromosome dose in said plurality of qualified samples;
based on said sequencing of said nucleic acid molecules in said test sample, calculating a test chromosome dose for said test chromosome of interest, wherein said calculating a test chromosome dose comprises determining a parameter for said test chromosome of interest and at least one normalizing test chromosome, and wherein said at least one normalizing test chromosome corresponds to said at least one normalizing chromosome sequence;
comparing said test chromosome dose to at least one threshold value; and
determining said fetal aneuploidy based on the outcome of step (f).
54 . The method of claim 53 , wherein said parameter for said qualified chromosome of interest and at least one qualified normalizing chromosome relates the number of sequence tags mapped to said qualified chromosome of interest to the number of tags mapped to said normalizing chromosome sequence, and wherein said parameter for said test chromosome of interest and at least one normalizing test chromosome relates the number of sequence tags mapped to said test chromosome of interest to the number of tags mapped to said normalizing chromosome sequence.
55 . The method of claim 53 , wherein said test and qualified sample is substantially cell-free biological sample.
56 . The method of claim 53 , wherein said sample is chosen from a maternal blood, plasma, serum, urine and saliva.
57 . The method of claim 53 , wherein said sample is a maternal plasma sample.
58 . The method of claim 53 , wherein said fetal and maternal nucleic acid molecules are cell-free DNA molecules.
59 . The method of claim 53 , wherein said sequencing is next generation sequencing (NGS).
60 . The method of claim 53 , wherein said sequencing is massively parallel sequencing using sequencing-by-synthesis with reversible dye terminators.
61 . The method of claim 53 , wherein said sequencing is sequencing-by-ligation.
62 . The method of claim 53 , wherein said step of sequencing comprises an amplification.
63 . The method of claim 53 , wherein said sequencing is single molecule sequencing.
64 . The method of claim 53 , wherein said chromosomal aneuploidy is chosen from trisomy 8, trisomy 13, trisomy 15, trisomy 16, trisomy 18, trisomy 21, trisomy 22, monosomy X, and XXX.
65 . The method of claim 53 , wherein said chromosome of interest is chosen from chromosome 8, chromosome 13, chromosome 15, chromosome 16, chromosome 18, chromosome 21, chromosome 22, and chromosome X.
66 . A method for identifying copy number variation (CNV) of a sequence of interest in a test sample comprising the steps of:
obtaining a test sample and a plurality of qualified samples, said test sample comprising test nucleic acid molecules and said plurality of qualified samples comprising qualified nucleic acid molecules;
sequencing at least a portion of said qualified and test nucleic acid molecules, wherein said sequencing comprises providing a plurality of mapped sequence tags for a test and a qualified sequence of interest, and for at least one test and at least one qualified normalizing sequence;
based on said sequencing of said qualified nucleic acid molecules, calculating a qualified sequence dose for said qualified sequence of interest in each of said plurality of qualified samples, wherein said calculating a qualified sequence dose comprises determining a parameter for said qualified sequence of interest and at least one qualified normalizing sequence;
based on said qualified sequence dose, identifying at least one qualified normalizing sequence, wherein said at least one qualified normalizing sequence has the smallest variability and/or the greatest differentiability in sequence dose in said plurality of qualified samples;
based on said sequencing of said nucleic acid molecules in said test sample, calculating a test sequence dose for said test sequence of interest, wherein said calculating a test sequence dose comprises determining a parameter for said test sequence of interest and at least one normalizing test sequence, and wherein said at least one normalizing test sequence corresponds to said at least one qualified normalizing sequence;
comparing said test sequence dose to at least one threshold value; and
identifying said copy number variation of said sequence of interest in said test sample based on the outcome of step (f).
67 . The method of claim 66 , wherein said parameter for said qualified sequence of interest and at least one qualified normalizing sequence relates the number of sequence tags mapped to said qualified sequence of interest to the number of tags mapped to said qualified normalizing sequence, and wherein said parameter for said test sequence of interest and said at least one normalizing test sequence relates the number of sequence tags mapped to said test sequence of interest to the number of tags mapped to said normalizing test sequence.
68 . The method of claim 66 , wherein said sequencing is next generation sequencing (NGS).
69 . The method of claim 66 , wherein said sequencing is massively parallel sequencing using sequencing-by-synthesis with reversible dye terminators.
70 . The method of claim 66 , wherein said sequencing is sequencing-by-ligation.
71 . The method of claim 66 , wherein said sequencing comprises an amplification.
72 . The method of claim 55 , wherein said sequencing is single molecule sequencing.
73 . The method of claim 66 , wherein said CNV of a sequence of interest is an aneuploidy.
74 . The method of claim 66 , wherein said aneuploidy is a chromosomal or a partial aneuploidy.
75 . The method of claim 74 , wherein said chromosomal aneuploidy selected from trisomy 8, trisomy 13, trisomy 15, trisomy 16, trisomy 18, trisomy 21, trisomy 22, monosomy X, and XXX.
76 . The method of claim 74 , wherein said partial aneuploidy is a partial deletion or a partial insertion.
77 . The method of claim 66 , wherein said test and qualified samples are biological fluid samples.
78 . The method of claim 66 , wherein said samples are plasma samples.
79 . The method of claim 66 , wherein said test and qualified sample is a plasma sample obtained from a pregnant human subject.
80 . The method of claim 66 , wherein said test and qualified sample is a plasma sample obtained from a subject that is known or is suspected of having cancer.
81 . The method of claim 1 , 14 , 27 , or 40 , wherein said maternal blood sample is a plasma sample.