# US 2019/0169681 A1 · Assay systems for genetic analysis > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2019/0169681 A1 - Document ID: US20190169681A1 - Status: Published application - Kind: A1 - Application number: 16/159,588 - Filed: 2018-10-12 - Published: 2019-06-06 - Earliest claimed: 2010-08-06 - Assignees: Ariosa Diagnostics, Inc. - Applicants: Ariosa Diagnostics, Inc. - Inventors: Andrew Sparks; Arnold Oliphant,; Jacob Zahn; Ken Song; John Stuelpnagel - Web page: https://patentgrep.com/patent/US20190169681A1 ## CPC - C12Q 1/6827: for detection of mutation or polymorphism - C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection - C12Q 1/6862: Ligase chain reaction [LCR] - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract The present invention provides assays systems and methods for detection of chromosomal abnormalities and status of single loci associated with monogenic or polygenic traits in a sample containing nucleic acids from a maternal and a fetal source. ## Claims ### Claim at position 1 -21. (canceled) ### Claim 22 A method for detection of the presence or absence of a fetal copy number variation (CNV) of a genomic region and determination of genetic status of one or more maternal and/or fetal loci of interest in a maternal plasma or serum sample comprising fetal and maternal cell-free DNA, the method comprising: (a) hybridizing (i) a first set of at least 24 pairs of fixed sequence oligonucleotides with (ii) fetal and maternal cell-free DNA in the maternal plasma or serum sample, wherein the oligonucleotides of each pair are complementary to regions in first non-polymorphic loci within a first nucleic acid region of interest of a first chromosome or a portion of the first chromosome, and wherein the melting temperatures (Tms) of first fixed sequence oligonucleotides of each of the pairs of the first set vary in a range of two degrees centigrade; (b) hybridizing (i) a second set of at least 24 pairs of fixed sequence oligonucleotides with (ii) the fetal and maternal cell-free DNA in the maternal plasma or serum sample, wherein the oligonucleotides of each pair are complementary to regions in second non-polymorphic loci within a second nucleic acid region of interest of a second chromosome or a portion of the second chromosome and wherein the Tms of first fixed sequence oligonucleotides of each of the pairs of the second set vary in a range of two degrees centigrade; (c) annealing bridging oligonucleotides to the first and second non-polymorphic loci, wherein the bridging oligonucleotides hybridize between and adjacent to the hybridized oligonucleotides of the first and second sets of fixed sequence oligonucleotide pairs to create adjacently hybridized oligonucleotides spanning the non-polymorphic loci in the first and second nucleic acid regions of interest; (d) ligating the adjacently hybridized bridging oligonucleotides and fixed sequence oligonucleotides to produce contiguous ligation products; (e) amplifying the contiguous ligation products to produce amplification products, and further amplifying one or more selected nucleic acid regions comprising the one or more loci of interest in the maternal and fetal cell-free DNA in the maternal plasma or serum sample with primers complementary to the selected nucleic acid regions comprising the one or more loci of interest to produce amplified selected maternal and fetal nucleic acid regions in the maternal plasma or serum sample; (f) detecting the amplification products to count the relative frequency of each non-polymorphic locus within the first and second nucleic acid regions of interest; and (g) detecting a fetal CNV of the first nucleic acid region of interest in the maternal serum or plasma sample relative to the second nucleic acid region of interest, and determining if the amplified selected maternal and fetal nucleic acid regions from the one or more loci of interest are detected in a frequency indicative of maternal and/or fetal status of the one or more loci of interest. ### Claim 23 (depends on 22) The method of claim 22, wherein the fetal CNV is a fetal chromosomal aneuploidy. ### Claim 24 (depends on 22) The method of claim 22, wherein the fixed sequence oligonucleotides of the first and second sets comprise universal primer regions. ### Claim 25 (depends on 24) The method of claim 24, wherein the universal primer regions are used in sequence determination of the amplification products. ### Claim 26 (depends on 24) The method of claim 24, wherein the fixed sequence oligonucleotides of each set comprises at least one universal primer region, wherein the at least one universal primer region of the first set of fixed sequence oligonucleotides differs from the at least one universal primer region of the second set of fixed sequence oligonucleotides. ### Claim 27 (depends on 22) The method of claim 22, wherein one or both of the fixed sequence oligonucleotides in each pair comprises at least one index. ### Claim 28 (depends on 22) The method of claim 22, wherein step (g) is performed by high throughput sequencing. ### Claim 29 (depends on 27) The method of claim 27, wherein the amplification products are detected by sequencing the at least one index. ### Claim 30 (depends on 27) The method of claim 27, wherein the at least one index comprises a locus index. ### Claim 31 (depends on 27) The method of claim 27, wherein the at least one index comprises both a sample index and a locus index, and wherein the sample index and the locus index are on different fixed sequence oligonucleotides in a pair. ### Claim 32 (depends on 22) The method of claim 22, wherein the amplification products are detected on an array. ### Claim 33 A method for detection of the presence or absence of a fetal chromosomal aneuploidy of a first chromosome and determination of genetic status of one or more maternal and/or fetal loci of interest in a maternal plasma or serum sample comprising fetal and maternal cell-free DNA, the method comprising: (a) hybridizing (i) a first set of at least 24 pairs of fixed sequence oligonucleotides with (ii) the fetal and maternal cell-free DNA in the maternal plasma or serum sample, wherein the oligonucleotides of each pair are complementary to regions in first non-polymorphic loci within a first chromosome or a portion of the first chromosome, and wherein the melting temperatures (Tms) of first fixed sequence oligonucleotides of each of the pairs of the first set vary in a range of two degrees centigrade; (b) hybridizing (i) a second set of at least 24 pairs of fixed sequence oligonucleotides with (ii) the fetal and maternal cell-free DNA in the maternal plasma or serum sample, wherein the oligonucleotides of each pair are complementary to regions in second non-polymorphic loci within a second chromosome or a portion of the second chromosome and wherein the Tms of first fixed sequence oligonucleotides of each of the pairs of the second set vary in a range of two degrees centigrade; (c) annealing bridging oligonucleotides to the first and second non-polymorphic loci, wherein the bridging oligonucleotides hybridize between and adjacent to the hybridized oligonucleotides of the first and second sets of fixed sequence oligonucleotide pairs to create adjacently hybridized oligonucleotides spanning the non-polymorphic loci in the first and second chromosomes of interest; (d) ligating the adjacently hybridized bridging oligonucleotides and fixed sequence oligonucleotides to produce contiguous ligation products; (e) amplifying the contiguous ligation products to produce amplification products, and further amplifying one or more selected nucleic acid regions comprising the one or more loci of interest in the maternal and fetal cell-free DNA in the maternal plasma or serum sample with primers complementary to the selected nucleic acid regions comprising the one or more loci of interest to produce amplified selected maternal and fetal nucleic acid regions in the maternal plasma or serum sample; (f) detecting the amplification products to count the relative frequency of each non-polymorphic locus within the first and second chromosomes of interest; and (g) detecting a fetal chromosomal aneuploidy of the first chromosome in the maternal serum or plasma sample if the relative frequencies of the first and second chromosome of interest vary statistically, and determining if the amplified selected maternal and fetal nucleic acid regions from the one or more loci of interest are detected in a frequency indicative of maternal and/or fetal status of the one or more loci of interest. ### Claim 34 (depends on 33) The method of claim 33, wherein the fixed sequence oligonucleotides of the first and second sets comprise universal primer regions. ### Claim 35 (depends on 34) The method of claim 34, wherein the universal primer regions are used in sequence determination of the amplification products. ### Claim 36 (depends on 33) The method of claim 33, wherein one or both of the fixed sequence oligonucleotides in each pair comprises at least one index. ### Claim 37 (depends on 36) The method of claim 36, wherein the amplification ligation products are detected by sequencing the at least one index. ### Claim 38 (depends on 36) The method of claim 36, wherein the at least one index comprises a locus index. ### Claim 39 (depends on 36) The method of claim 36, wherein the at least one index comprises a sample index. ### Claim 40 (depends on 36) The method of claim 36, wherein the at least one index comprises both a sample index and a locus index, and wherein the sample index and the locus index are present on the same fixed oligonucleotide in a set. ### Claim 41 (depends on 36) The method of claim 36, wherein the at least one index comprises both a sample index and a locus index, and wherein the sample index and the locus index are on different fixed sequence oligonucleotides within a pair. ### Claim 42 (depends on 33) The method of claim 33, wherein the amplification products are detected on an array. ### Claim 43 A method for detection of the presence or absence of a fetal chromosomal aneuploidy of a first chromosome and determination of genetic status of one or more maternal and/or fetal loci of interest in a maternal plasma or serum sample comprising fetal and maternal cell-free DNA, the method comprising: (a) hybridizing (i) a first set of at least 24 pairs of fixed sequence oligonucleotides with (ii) the fetal and maternal cell-free DNA in the maternal plasma or serum sample, wherein the oligonucleotides of each pair are complementary to regions in first non-polymorphic loci within a first chromosome or a portion of the first chromosome, wherein the melting temperatures (Tms) of first fixed sequence oligonucleotides of each of the pairs of the first set vary in a range of two degrees centigrade, and wherein at least one of the fixed sequence oligonucleotides in each pair of the first set of fixed sequence oligonucleotides comprises a locus index; (b) hybridizing (i) a second set of at least 24 pairs of fixed sequence oligonucleotides with (ii) the fetal and maternal cell-free DNA in the maternal plasma or serum sample, wherein the oligonucleotides of each pair are complementary to regions in second non-polymorphic loci within a second chromosome or a portion of the second chromosome, wherein the Tms of first fixed sequence oligonucleotides of each of the pairs of the second set vary in a range of two degrees centigrade, and wherein at least one of the fixed sequence oligonucleotides in each pair of the second set of fixed sequence oligonucleotides comprises a locus index; (c) annealing bridging oligonucleotides to the first and second non-polymorphic loci, wherein the bridging oligonucleotides hybridize between and adjacent to the hybridized oligonucleotides of the first and second sets of fixed sequence oligonucleotide pairs to create adjacently hybridized oligonucleotides spanning the non-polymorphic loci in the first and second chromosomes of interest; (d) ligating the adjacently hybridized bridging oligonucleotides and fixed sequence oligonucleotides to produce contiguous ligation products; (e) amplifying the contiguous ligation products to produce amplification products, and further amplifying one or more selected nucleic acid regions comprising the one or more loci of interest in the maternal and fetal cell-free DNA in the maternal plasma or serum sample with primers complementary to the selected nucleic acid regions comprising the one or more loci of interest to produce amplified selected maternal and fetal nucleic acid regions in the maternal plasma or serum sample; (f) detecting the amplification products to count the relative frequency of each non-polymorphic locus within the first and second chromosomes of interest; and (g) detecting a fetal chromosomal aneuploidy of the first chromosome in the maternal serum or plasma sample if the relative frequencies of the first and second chromosome of interest vary statistically, and determining if the amplified selected maternal and fetal nucleic acid regions from the one or more loci of interest are detected in a frequency indicative of maternal and/or fetal status of the one or more loci of interest. ### Claim 44 (depends on 43) The method of claim 43, wherein the amplification products are detected on an array. ### Claim 45 (depends on 44) The method of claim 44, wherein the amplification products are detected on an array via the locus index.