Granted Patent B2

US 9,567,639 · App. 14/453,396 · Granted Feb 14, 2017

Inventors: Arnold Oliphant (San Jose, CA); Jacob Zahn (San Jose, CA); Kara Juneau (San Jose, CA); Patrick Bogard (San Jose, CA); Stephanie Huang (San Jose, CA)

Assignee: Ariosa Diagnostics, Inc.

C12Q1/6883C12Q1/6827C12Q1/6837C12Q1/6862G06F19/20C12Q2600/166

Abstract

Claims (75)

1. An assay method for providing a statistical likelihood of a fetal copy number variation comprising:

providing a maternal plasma or serum sample comprising maternal and fetal cell free DNA;

interrogating at least 48 non-polymorphic loci from a first target genomic region by hybridizing sets of at least two fixed sequence oligonucleotides comprising a region complementary to a locus in the first target genomic region, wherein one of the fixed sequence oligonucleotides of each set comprises a first capture region, a first label binding region, and two restriction sites;

interrogating at least 48 non-polymorphic loci from a second target genomic region by hybridizing sets of at least two fixed sequence oligonucleotides comprising a region complementary to a locus in the second target genomic region, wherein one of the fixed sequence oligonucleotides of each set comprises the first capture region, a second label binding region, and two restriction sites;

ligating the hybridized fixed sequence oligonucleotides;

amplifying the ligated fixed sequence oligonucleotides to create amplicons;

cleaving the amplicons at the restriction sites to create cleaved amplicons, wherein each cleaved amplicon comprises the first capture region and the first or second label binding region;

detecting the cleaved amplicons from the first and second target genomic regions via hybridization of the first capture regions of the cleaved amplicons to an array comprising capture probes complementary to the first capture regions, wherein the cleaved amplicons from the first and second target genomic regions hybridize competitively to the capture probes complementary to the first capture regions;

quantifying the capture regions of the cleaved amplicons to determine a relative frequency of the interrogated non-polymorphic loci from the first and second target genomic regions by detecting the first and second label binding regions;

estimating the relative frequency of the first and second target genomic regions based on the determined relative frequency of the first and second label binding regions;

interrogating at least 48 polymorphic loci from at least one target genomic region different from the first and second target genomic regions by hybridizing sets of at least three fixed sequence allele-specific oligonucleotides for each polymorphic locus, wherein two of the at least three allele-specific oligonucleotides of each set comprises a sequence complementary to one allele at a polymorphic locus, a capture region specific for each polymorphic locus, a different label binding region for each allele at the polymorphic locus, and two restriction sites;

ligating the hybridized fixed sequence allele-specific oligonucleotides;

amplifying the ligated fixed sequence allele-specific oligonucleotides to create allele-specific amplicons;

cleaving the allele-specific amplicons at the restriction sites to create cleaved allele-specific amplicons, wherein each cleaved allele-specific amplicon comprises a polymorphic locus-specific capture region and an allele-specific label binding region;

detecting the cleaved allele-specific amplicons from the polymorphic loci via competitive hybridization of the polymorphic locus-specific capture regions of the cleaved allele-specific amplicons to capture regions on the array;

quantifying the alleles of the polymorphic loci by detecting the allele-specific label binding regions for each allele on the cleaved allele-specific amplicons to determine the fraction of fetal DNA in the sample;

determining the fraction of fetal DNA; and

calculating a statistical likelihood of a fetal copy number variation in the maternal sample using the estimated relative frequency of the first and second target genomic regions in the sample and the fraction of fetal DNA.

2. The assay method of claim 1 , wherein three or more fixed sequence oligonucleotides for each set are used to interrogate each non-polymorphic locus in the first and second target genomic regions.

3. The assay method of claim 1 , wherein each amplifying step uses universal amplification through a polymerase chain reaction.

4. The assay method of claim 1 , further comprising:

identifying low frequency alleles from the quantified alleles of the polymorphic loci where the maternal DNA is homozygous and the fetal DNA is heterozygous; and

using the low frequency alleles from the polymorphic loci to determine the fraction of fetal DNA.

5. The assay method of claim 1 , wherein the first genomic region is a chromosome.

6. The assay method of claim 1 , wherein the second genomic region is a chromosome.

7. The assay method claim 1 , wherein the first genomic region is a sub-chromosomal region.

8. The assay method claim 1 , wherein the second genomic region is a sub-chromosomal region.

9. The assay method of claim 1 , wherein the label binding regions of the fixed sequence oligonucleotides each comprise a label, wherein the first label binding regions each comprise a first label, and the second label binding regions each comprise a second label.

10. The assay method of claim 1 , wherein the label binding regions of the fixed sequence oligonucleotides each comprise a sequence complementary to an oligonucleotide comprising a label, wherein the first label binding regions each comprise a sequence complementary to a first oligonucleotide comprising a first label, and the second label binding regions each comprise a sequence complementary to a second oligonucleotide comprising a second label.

11. An assay method for determining a likelihood of a fetal aneuploidy comprising the steps of:

providing a maternal plasma or serum sample comprising maternal and fetal cell free DNA;

introducing at least fifty first sets of two or more fixed sequence oligonucleotides complementary to a non-polymorphic locus in a first target genomic region in the maternal sample under conditions that allow a complementary region of each fixed sequence oligonucleotide to specifically hybridize to the non-polymorphic locus, wherein at least one of the fixed sequence oligonucleotides of each set comprises a universal primer site, a first capture region, a first label binding region, and two restriction sites;

introducing at least fifty second sets of two or more fixed sequence oligonucleotides complementary to a non-polymorphic locus in a second target genomic region in the maternal sample under conditions that allow a complementary region of each fixed sequence oligonucleotide to specifically hybridize to the non-polymorphic locus, wherein at least one of the fixed sequence oligonucleotides of each set comprises a universal primer site, the first capture region, a second label binding region, and two restriction sites;

introducing at least fifty third sets of three or more fixed sequence oligonucleotides complementary to a set of polymorphic loci in the maternal sample under conditions that allow a complementary region of each fixed sequence oligonucleotide to specifically hybridize to a polymorphic locus, wherein at least two of the three fixed sequence oligonucleotides of each set comprises a universal primer site, a sequence complementary to one allele at a polymorphic locus, an allele-specific label binding region for each allele at the polymorphic locus, two restriction sites, and a polymorphic locus-specific capture region, wherein the capture region for each polymorphic locus is different from the capture region for every other polymorphic locus and different from the first capture region;

hybridizing the first, second and third sets of fixed sequence oligonucleotides to the first and second target genomic regions and the polymorphic loci;

extending at least one of the hybridized fixed sequence oligonucleotides of the first, second and third sets to form adjacently hybridized fixed sequence oligonucleotides;

ligating the hybridized fixed sequence oligonucleotides of the first, second and third sets to create ligation products;

amplifying the ligation products using the universal primer sites to create amplicons corresponding to the polymorphic loci;

cleaving the amplicons at the restriction sites to create cleaved amplicons, wherein each cleaved amplicon comprises one capture region and one label binding region;

applying the cleaved amplicons to an array, wherein the array comprises first capture probes complementary to the first capture regions on the cleaved amplicons from the first and second target genomic regions, and wherein the array comprises capture probes complementary to the capture regions on the cleaved amplicons from each polymorphic locus;

hybridizing the first capture regions of the cleaved amplicons from the first and second target genomic regions to first capture probes on an array;

hybridizing the capture regions of the cleaved amplicons from the polymorphic loci to capture probes on the array;

detecting the hybridized cleaved amplicons;

quantifying a relative frequency of the cleaved amplicons corresponding to loci from the first target genomic region and a relative frequency of the cleaved amplicons corresponding to loci from the second target genomic region by detecting the first and second label binding regions;

quantifying a relative frequency of each allele from the polymorphic loci by detecting the allele-specific label binding regions for each allele on the cleaved amplicons to determine a percent fetal cell free DNA; and

computing a likelihood of fetal aneuploidy using the relative frequency of the cleaved amplicons corresponding to loci from the first and second target genomic regions to determine the likelihood of a fetal aneuploidy and the determined percent fetal cell free DNA.

12. The assay method of claim 11 , wherein the relative frequencies of the cleaved amplicons corresponding to the loci from the first target genomic region are summed and the relative frequencies of the cleaved amplicons corresponding to the loci from the second target genomic region are summed, and the sum from the cleaved amplicons corresponding to the loci from the first target genomic region are compared to the sum from the cleaved amplicons corresponding to the loci from the second target genomic region to calculate a target genomic region ratio.

13. The assay method of claim 11 , wherein a single hybridized fixed sequence oligonucleotide of each set is extended to form adjacently hybridized fixed sequence oligonucleotides.

14. The assay method of claim 11 , wherein the label binding regions of the fixed sequence oligonucleotides each comprise a label, wherein the first label binding regions each comprise a first label, and the second label binding regions each comprise a second label.

15. The assay method of claim 11 , wherein the label binding regions of the fixed sequence oligonucleotides each comprise a sequence complementary to an oligonucleotide comprising a label, wherein the first label binding regions each comprise a sequence complementary to a first oligonucleotide comprising a first label, and the second label binding regions each comprise a sequence complementary to a second oligonucleotide comprising a second label.

16. An assay method for determining a likelihood of a fetal aneuploidy comprising the steps of:

providing a maternal plasma or serum sample comprising maternal and fetal cell free DNA;

introducing at least fifty first sets of two or more fixed sequence oligonucleotides complementary to a set of non-polymorphic loci in a first target genomic region in the maternal sample under conditions that allow a complementary region of each fixed sequence oligonucleotide to specifically hybridize to the non-polymorphic loci, wherein at least one of the fixed sequence oligonucleotides of each set comprises a universal primer site, a first capture region, a first label binding region, and two restriction sites;

introducing at least fifty second sets of two or more fixed sequence oligonucleotides complementary to a set of non-polymorphic loci in a second target genomic region in the maternal sample under conditions that allow a complementary region of each fixed sequence oligonucleotide to specifically hybridize to the non-polymorphic loci, wherein at least one of the fixed sequence oligonucleotides of each set comprises a universal primer site, a first capture region, a second label binding region, and two restriction sites;

introducing two or more third sets of three or more fixed sequence oligonucleotides complementary to a set of polymorphic loci in the maternal sample under conditions that allow a complementary region of each fixed sequence oligonucleotide to specifically hybridize to a polymorphic locus, wherein at least two of the three or more fixed sequence oligonucleotides of each set comprises a universal primer site, a sequence complementary to one allele at the polymorphic locus, an allele-specific label binding region for each allele at the polymorphic locus, two restriction sites, and a polymorphic locus-specific capture region, wherein the capture region for each polymorphic locus is different from the capture region for every other polymorphic locus and different from the first capture region;

hybridizing the first, second and third sets of fixed sequence oligonucleotides to the first and second target genomic regions and polymorphic loci;

extending at least one of the hybridized fixed sequence oligonucleotides of the first, second and third sets to form adjacently hybridized fixed sequence oligonucleotides for each set;

ligating the adjacently hybridized fixed sequence oligonucleotides from the first, second and third sets to create ligation products;

amplifying the ligation products using the universal primer sites to create amplicons;

cleaving the amplicons at the restriction sites to create cleaved amplicons, wherein each cleaved amplicon comprises one capture region and one label binding region;

applying the cleaved amplicons to an array, wherein the array comprises first capture probes complementary to the first capture regions on the cleaved amplicons from the first and second target genomic regions, and wherein the array comprises capture probes complementary to the capture regions on the cleaved amplicons from each polymorphic locus;

hybridizing the first capture regions of the cleaved amplicons from the first and second target genomic regions to first capture probes on an array;

hybridizing the capture regions of the cleaved amplicons from the polymorphic loci to capture probes on the array;

detecting the hybridized cleaved amplicons;

quantifying a relative frequency of each allele from the polymorphic loci by detecting the allele-specific label binding regions for each allele on the cleaved amplicons to determine a percent fetal cell free DNA;

determining the percent of fetal cell free DNA by identifying low frequency alleles from the quantified alleles where a maternal locus is homozygous and a corresponding fetal locus is heterozygous;

quantifying a relative frequency of cleaved amplicons corresponding to loci from the first target genomic region and a relative frequency of cleaved amplicons corresponding to loci from the second target genomic region by detecting the first and second label binding regions; and

computing a likelihood of a fetal aneuploidy using the relative frequency of cleaved amplicons corresponding to loci from the first and second target genomic regions and the percent fetal cell free DNA.

17. The assay method of claim 16 , further comprising comparing the relative frequencies of the cleaved amplicons corresponding to loci from the first and second target genomic regions and adjusting the likelihood of a fetal aneuploidy based on the percent fetal cell-free DNA.

18. The assay method of claim 16 wherein the relative frequencies of the cleaved amplicons corresponding to the loci from the first target genomic region are summed and the relative frequencies of the cleaved amplicons corresponding to the loci from the second target genomic region are summed, and the sum from the cleaved amplicons corresponding to the loci from the first target genomic region are compared to the sum from the cleaved amplicons corresponding to the loci from the second target genomic region to calculate a target genomic region ratio.

19. The assay method of claim 16 , wherein the polymorphic loci are autosomal loci.

20. The assay method of claim 16 , wherein one or more corresponding fetal polymorphic loci comprise more than one genetic variation as compared to a maternal locus.

21. The assay method of claim 16 , wherein the cleaved amplicons from loci of the first and second target genomic regions and the cleaved amplicons from the polymorphic loci are created in a single vessel.

22. The assay method of claim 16 , wherein the label binding regions of the fixed sequence oligonucleotides each comprise a label, wherein the first label binding regions each comprise a first label, and the second label binding regions each comprise a second label.

23. The assay method of claim 16 , wherein the label binding regions of the fixed sequence oligonucleotides each comprise a sequence complementary to an oligonucleotide comprising a label, wherein the first label binding regions each comprise a sequence complementary to a first oligonucleotide comprising a first label, and the second label binding regions each comprise a sequence complementary to a second oligonucleotide comprising a second label.

Assignments (3)

Continuity (8)