(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`3 June 2011 (03.06.2011) (10) International Publication Number
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`(43) International Publication Date
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`WO 2011/066476 A1
`
`
`(51)
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`(21)
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`(22)
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`(25)
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`(26)
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`(30)
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`International Patent Classification:
`C12P 19/34 (2006.01)
`
`International Application Number:
`PCT/US2010/058124
`
`International Filing Date:
`25 November 2010 (25.11.2010)
`
`Filing Language:
`
`Publication Language:
`
`Priority Data:
`61/264,591
`25 November 2009 (25.11.2009)
`61/309,837
`2 March 2010 (02.03.2010)
`61/309,845
`2 March 2010 (02.03.2010)
`61/317,635
`25 March 2010 (25.03.2010)
`61/317,639
`25 March 2010 (25.03.2010)
`61/317,684
`25 March 2010 (25.03.2010)
`61/341,065
`25 March 2010 (25.03.2010)
`61/341,218
`25 March 2010 (25.03.2010)
`61/380,981
`8 September 2010 (08.09.2010)
`61/409,106
`1 November 2010 (01.11.2010)
`61/109,473
`2 November 2010 (02.11.2010)
`61/410,769
`5 November 2010 (05.11.2010)
`
`English
`
`English
`
`US
`US
`US
`US
`US
`US
`US
`US
`US
`US
`US
`US
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`(71)
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`(72)
`(75)
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`Applicant (for all designated States except US): QUAN-
`TALIFE, INC.
`[US/US]; 7068 K011 Center Parkway,
`Suite 401, Pleasanton, CA 94566 (US).
`
`Inventors; and
`Inventors/Applicants (for US only): HINDSON, Ben-
`jamin [AU/US]; 1039 Bannock Strcct, Livcrmorc, CA
`94551 (US). SAXONOV, Serge [US/US]; 10 De Anza
`Cort, San Mateo, CA 94402 (US). BELGRADER, Philip
`[US/US]; 89 Robinson Landing Road, Sevena Park, MD
`21146 (US). NESS, Kevin [CA/US]; 24 Baytree Way,
`Pat. #10, San Mateo, CA 94402 (US). LUCERO,
`
`(74)
`
`(81)
`
`Michael [US/US]; 634 Pine Terrace, South San Francis-
`co, CA 94080 (US). COLSTON, Billy [US/US]; 9981
`Torreon Ave, San Ramon, CA 94583 (US).
`
`Agents: STOPAK, Kimberly, S. et al.; Wilson Sonsini
`Goodrich & Rosati, 650 Page Mill Road, Palo Alto, CA
`94304-1050 (US).
`
`Designated States (unless otherwise indicated, for every
`kind ofnational protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ,
`CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO,
`DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI,
`NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD,
`SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR,
`TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(84)
`
`Pub
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG,
`ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, IIR, IIU, IE, IS, IT, LT, LU,
`LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK,
`SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, ML, MR, NE, SN, TD, TG).
`lished:
`
`with international search report (Art. 21(3))
`
`before the expiration of the time limit for amending the
`claims and to be republished in the event of receipt of
`amendments (Rule 48.2(h))
`
`(54) Title: METHODS AND COMPOSITIONS FOR DETECTING GENETIC MATERIAL
`
`(57) Abstract: This invention provides compositions and methods for detecting differences in copy number of a target polynu—
`cleotide. In some cases, the methods and compositions provided herein are usefiil for diagnosis of fetal genetic abnormalities,
`when the starting sample is maternal tissue (e. g., blood, plasma). The methods and materials described apply techniques for allow—
`ing detection of small, but statistically significant, differences in polynucleotide copy number.
`
`
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`WO 2011/066476
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`PCT/US2010/058124
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`METHODS AND COMPOSITIONS FOR DETECTING GENETIC MATERIAL
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`CROSS—REFERENCE TO RELATED APPLICATIONS
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`[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.
`
`61/264,591, filed on November 25, 2009; U.S. Provisional Application No. 61/309,837, filed on March 2, 2010;
`
`U.S. Provisional Application No. 61/309,845, filed on March 2, 2010; U.S. Provisional Application No. 61/317,635,
`
`filed on March 25, 2010; U.S. Provisional Application No. 61/317,639, filed on March 25, 2010; U.S. Provisional
`
`Application No. 61/317,684, filed on March 25, 2010; U.S. Provisional Application No. 61/341,065, filed on March
`
`25, 2010; U.S. Provisional Application No. 61/341,218, filed on March 25, 2010; U.S. Provisional Application No.
`
`61/3 80,981, filed on September 8, 2010; U.S. Provisional Application No. 61/409,106, filed on November 1, 2010;
`
`U.S. Provisional Application No. 61/409,473, filed on November 2, 2010; and U.S. Provisional Application No.
`
`61/410,769, filed on November 5, 2010, each of which is incorporated herein by reference in its entirety
`
`BACKGROUND OF THE INVENTION
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`[0001] Fetal aneuploidies are aberrations in chromosome number and commonly arise as a result of a meiotic
`
`nondisjunction during oogenesis or spermatogenesis; however, certain aneuploidies, such as trisomy 8, result more
`
`often from postzygotic mitotic disjunction micolaidis & Petersen (1998) Human Reproduction 13:313-319). Such
`aberrations include both reductions and increases in the normal chromosome number and can involve autosomes as
`
`well as the sex chromosomes. An example of a reduction aneuploidy is Turner's syndrome, which is typified by the
`
`presence of a single X sex chromosome. Examples of increases in chromosome number include Down's syndrome
`
`(trisomy of chromosome 21), Patau syndrome (trisomy of chromosome 13), Edwards syndrome (trisomy of
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`chromosome 18), and Kleinfelter's syndrome (an XXY trisomy of the sex chromosomes). Aneuploidies commonly
`
`lead to significant physical and neurological impairments which result in a large percentage of affected individuals
`
`failing to reach adulthood. In fact, fetuses having an autosomal aneuploidy involving a chromosome other than 13,
`
`18, or 21 generally die in utero. However, certain aneuploidies, such as Kleinfelter's syndrome, present far less
`
`pronounced phenotypes and those affected with other trisomies, such as XXY & XXX, often will mature to be
`
`fertile adults. In some cases, partial aneuploidy resulting in an abnormal copy number of a portion of a
`
`chromosome may result from an imbalanced nondisjunction.
`
`[0002] Prenatal diagnosis of fetal aneuploidies using invasive testing by amniocentesis or Chorionic Villus
`
`Sampling (CVS), are associated with a 0.5% to 2% procedure-related risk of pregnancy loss (D'Alton, M. E., (1994)
`
`Semin Perinatol 18: 140-62; Caughey AB (2006) Obstet Gynecol 108:612-6).
`
`[0003] Another barrier to accurately screening fetal aneuploidy is the low concentration of fetal DNA in maternal
`
`plasma, particularly at earlier gestational ages. Single or low multiplex assay approaches are unlikely to provide
`
`enough target counts to differentiate between an aneupoloid fetus (e. g., trisomy of chromosome 21) from a euploid
`
`fetus. There is also, generally, a need in the art for methods and compositions for detecting copy number variations
`
`in biological samples, not necessarily from maternal blood.
`
`SUMMARY OF THE INVENTION
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`[0004] The present disclosure provides methods and compositions for detecting copy number of a target
`
`polynucleotide within a population of genetic material. Partitioning may be used to subdivide the target
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`WO 2011/066476
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`polynucleotide into a plurality ofreaction volumes. In some cases, a probe to the target polynucleotide is
`
`subdivided into a plurality of reaction volumes.
`
`[0005]
`
`In some cases, the methods comprise the following steps: a. binding a first ligation probe to a first target
`
`polynucleotide;
`
`b. binding a second ligation probe to a second target polynucleotide; c.
`
`subjecting said first and
`
`second ligation probes to a ligation reaction in order to obtain one or more ligated products; d. partitioning said
`
`one or more ligated products into two or more partitions; e. amplifying a sequence within said one or more ligated
`
`products to obtain amplified products;
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`f. determining a number of said partitions that contain said amplified
`
`products; and g.
`
`calculating a copy number of said first target polynucleotide. In some cases, the target
`
`polynucleotide is not partitioned into said two or more partitions.
`
`[0006] Partitions can include a wide variety of types of partitions, including solid partitions (e. g., wells, tubes,
`
`etc.) and fluid partitions (e. g., aqueous droplets within an oil phase, such as a continuous oil phase, or aqueous
`
`droplets within a mixture of at least two immiscible fluids). The partitions may also be stable or unstable. For
`
`example, in some cases, during the amplification process said two or more partitions remain substantially intact. In
`
`some cases, the partitions are aqueous droplets Within an oil phase and said aqueous droplets remain substantially
`
`intact during the amplification reaction of the instant methods. The partitions (e. g., said aqueous droplets) may
`
`also remain substantially intact during the determination steps, when partitions are evaluated for the presence of
`
`one or more target polynucleotides (or probes to said polynucleotides). The partitions may comprise an
`
`amplification reaction that is initiated from said ligated product.
`
`[0007] The first and second ligation probes may bind (or be designed to bind) a variety of target polynucleotides;
`
`often a first ligation probe binds a first target polynucleotide and a second ligation probe binds a second
`
`polynucleotide. In some cases, the first and second ligation probes are each designed to bind to said first target
`
`polynucleotide. In other cases, said first ligation probe binds a first target polynucleotide that has a sequence that
`
`differs from the sequence of said second target poynucleotide.
`
`In some cases, first ligation probe is designed to
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`bind to a polynucleotide sequence that is conserved between individuals within a species. In some cases, first
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`ligation probe is designed to bind to a polynucleotide sequence that is conserved across two or more different
`
`species. In some cases, a ligation probe binds to a nonpolymorphic region of a chromosome.
`
`[0008]
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`In some embodiments, the method comprises ligating multiple ligation probes to said first target
`
`polynucleotide. For example, the method may comprise binding at least four ligation probes to said first target
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`polynucleotide. In other cases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 500, 1000,
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`5000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 100,000, 2,000,000, 3, 000,000, 4,000,000,
`
`5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000 or 10,000,000 ligation probes are used in the methods
`
`provided herein. Often, one or more of said ligation probes bind to a different polynucleotide (e. g., different
`
`chromosomes, different regions within the same chromosome). In some cases, a plurality of first ligation probes
`
`(e. g., target ligation probes) are used and a plurality of second ligation probes (e. g., reference ligation probes) are
`
`used in the present methods and compositions. The methods may further comprise binding at least four ligation
`
`probes to said first target polynucleotide and at least four ligation probes to said second target polynucleotide. In
`
`some cases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 500, 1000, 5000, 10,000,
`
`20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 100,000, 2,000,000, 3, 000,000, 4,000,000, 5,000,000, 6,000,000,
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`7,000,000, 8,000,000, 9,000,000 or 10,000,000 ligation probes are bound to said first or said second target
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`polynucleotide.
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`[0009] The first ligation probe may bind (or be designed to bind) to a first region within said first target
`
`polynucleotide and said second ligation probe may bind (or be designed to bind) to a second region within said first
`
`target polynucleotide, wherein said first and second regions do not have identical sequences.
`
`[0010] Often the first target polynucleotide is not identical to said second target polynucleotide. In some cases the
`
`first target polynucleotide is identical to the second target polynucleotide.
`
`In some examples, said first target
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`polynucleotide is a test chromosome and said second target polynucleotide is a reference chromosome. Examples of
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`test chromosomes include but are not limited to: chromosome 21, chromosome 13, chromosome 18, and the X
`
`chromosome. Said test chromosome may also be from the group consisting of chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9,
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`10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, and Y. A first target polynucleotide may be a segment ofa
`
`chromosome, such as a segment of a chromosome that is associated with fetal aneuploidy (either the chromosome
`
`or the segment may be associated with fetal aneuploidy).
`
`[0011] The methods and compositions provided herein often relate to ligating a probe to itself, ligating two probes
`
`together, and/or ligation products of said ligation reactions. Said ligation reactions may result in the ligation of a
`
`5’ region of said first ligation probe to a 3’ region of said first ligation probe to obtain a circular ligated product.
`
`In some cases, a ligation reaction results in the ligation of the 5’ region of said first ligation probe to the 3 ’ region
`
`of said second ligation probe, in order to obtain a linear ligated product comprising at least a portion of said first
`
`and second ligation probes. Said 5’ region and said 3’ region of said first ligation probe may each bind (or be
`
`designed to bind) adjacent sequences within said first target polynucleotide. Said adjacent sequences are separated
`
`by 0 nucleotides. Said 5’ region and said 3 ’ region of said first ligation probe may bind, or be designed to bind,
`
`neighboring sequences within said first target polynucleotide. Said neighboring sequences may be separated by at
`
`least one nucleotide. In some cases, the neighboring sequences are separated by a gap of at least 5, 10, 20, 30, 40
`
`,50, 100, 200, 300, 400, or 500 nucleotides.
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`[0012] The ligation reaction may further comprise a template-driven gap fill reaction to incorporate nucleotides in
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`the gap between said 5’ region and said 3’ region of said first ligation probe (or ofsaid second ligation probe).
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`[0013] The ligation probes may comprise a site cleavable by an enzyme. For example, the site cleavable by an
`
`enzyme may comprise one or more uracils. The uracils may be separated by other nucleotides in some cases. The
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`site cleavable by an enzyme may comprise a restriction site. The first ligation probe may be of a specific type, such
`
`as a molecular inversion probe, a padlock probe, a linear ligation probe, etc..
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`[0014] The methods provided herein may further comprise performing an enzymatic reaction to remove linear
`
`polynucleotides or single-stranded polynucleotides or double-stranded polynucleotides. For example, an
`
`exonuclease (e. g., Exo I, II, and/or III) may be used in the methods described herein. Often, exonuclease treatment
`
`removes all, or a substantial amount, of unbound ligation probes from a sample volume.
`
`[0015] The probes provided herein may be conjugated to signaling agent. Said first ligation probe may be
`
`conjugated to a first signaling agent and a second ligation probe is conjugated a second signaling agent. Often, a
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`plurality of such first and second ligation probes are used in the methods and compositions herein, wherein said
`
`probes are conjugated to the same signaling agent (e.g., identical fiuorophore) or to different signaling agents (e.g.,
`
`fluoropliores of different colors). Said first signaling agent may be a fluorescent marker of a first color and said
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`second signaling agent may be a fluorescent marker of a second color.
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`[0016] Detection of ligation probes is also often a step in the methods provided herein. The methods may
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`comprise detecting said first ligation probe with a first signaling agent and detecting said second ligation probe with
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`a second signaling agent. Said first ligation probe may comprise a first plurality ofligation probes, wherein each
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`probe within said plurality is directed to a different region of a first chromosome, and wherein said second ligation
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`probe comprises a second plurality of ligation probes, wherein each probe within said plurality is directed to a
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`different region of a second chromosome. In some cases, said first target polynucleotide is a test chromosome and
`
`said second target polynucleotide is a reference chromosome. In some cases, said first and second ligation probes
`
`are conjugated to the same color.
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`[0017] The methods and compositions provided herein may also involve a method of detecting copy number of a
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`target polynucleotide within a population of genetic material comprising: a. binding a first ligation probe to a first
`
`target polynucleotide;
`
`b. binding a second ligation probe to a second target polynucleotide; c.
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`subjecting said
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`first and second ligation probes to a ligation reaction in order to obtain one or more ligated products; d.
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`partitioning said one or more ligated products into two or more aqueous droplets within a continuous oil phase;
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`amplifying a sequence within said one or more ligated products to obtain amplified products; determining a
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`number of said two or more aqueous droplets that contain said amplified products; and g. calculating a copy
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`number of said target polynucleotide based on said number. In some cases, said target polynucleotide is not
`
`partitioned into said two or more aqueous droplets. In some cases, said target polynucleotide is not amplified.
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`In
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`some cases, or during said amplifying or determining steps, said two or more aqueous droplets remain
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`substantially intact.
`
`[0018]
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`In some cases, said two or more aqueous droplets comprise on average more than one ligated probe and
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`said method further comprises using an algorithm to calculate an average number of target ligated probes per
`
`aqueous droplet. Said two or more aqueous droplets may be greater than 4,000 droplets. In some cases, said two or
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`more aqueous droplets may be greater than 1,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, or
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`5,000,000 droplets.
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`[0019]
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`In some cases, said droplets are present in a single chamber at a high droplet/ml density. The density may
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`be greater than 100,000 aqueous droplets/ml. Examples of densities of droplets in a single chamber include: 10,000
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`droplets/mL, 100,000droplets/mL, 200,000droplets/mL, 300,000droplets/mL, 400,000droplets/mL,
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`500,000droplets/mL, 600,000droplets/mL, 700,000droplets/mL, 800,000droplets/mL, 900,000droplets/mL or
`
`1,000,000drop1ets/mL. The droplets used in any of the methods or compositions provided herein may be
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`monodisperse droplets. The droplets may have, on average, a diameter of between 50 nm and 300 mm. In some
`
`embodiments, the droplet diameter may be, on average, about .001, .01, .05, .1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80,
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`100, 120, 130, 140, 150, 160, 180, 200, 300, 400, or 500 microns In some cases,
`
`the droplets do not comprise a
`
`substantial number of beads conjugated to oligonucleotides.
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`[0020] The aqueous droplets may be present within an oil fluid or phase. The oil phase may comprise an anionic
`
`flourosurfactant an ammonium salt of an anionic fluorosurfactant, such as KrytoxTM. Krytox may be selected from
`
`a group consisting of Krytox AS, Krytox FSH, and morpholino derivative of Krytox FSH. The oil phase may
`
`comprise a fluorinated oil.
`
`[0021] The methods provided herein (e.g., detecting copy number using droplets) can be used to detect said first
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`target polynucleotide within a population of genetic material comprising less than 1,000 copies of said first target
`
`polynucleotide. In some cases, said two or more aqueous droplets comprise on average more than one ligated
`
`probe and said method further comprises using an algorithm to calculate an average number of target ligated probes
`
`per aqueous droplet.
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`[0022] The droplets may comprise a first target polynucleotide that is a chromosomal segment associated with a
`
`genetic disorder. The droplets may comprise a specific type of ligation probe (e. g., padlock probe, molecular
`
`inversion probe, ligation detection reaction (LDR) probe, etc.). The ligation probe may be subjected to a ligation
`
`reaction that ligates the 5’ region of the ligation probe to the 3 ’ region of the ligation probe.
`
`[0023] The methods and compositions provided herein may also relate to a method of detecting a fetal genetic
`
`condition comprising: a. obtaining a mixture of maternal and fetal genetic material comprising target
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`polynucleotides; b. combining said mixture with targeting oligonucleotides that bind said target polynucleotides;c.
`
`subdividing said targeting oligonucleotides into reaction volumes, wherein at least one of said reaction volumes
`
`comprises no target polynucleotide and no targeting oligonucleotide; d. performing an amplification reaction within
`
`said reaction volumes; e. detecting the presence of said target polynucleotide or said targeting oligonucleotide
`
`within said reaction volumes; and
`
`f. determining the relative level of said target polynucleotide in said mixture in order to detect a fetal genetic
`condition.
`
`[0024] The reaction volumes may be aqueous droplets within a continuous oil phase. The targeting
`
`oligonucleotides may comprise one or more primer pairs;
`
`ligation probes; molecular inversion probes; ligation
`
`detection reaction (LDR) probes; padlock probes; and any combination thereof. The reaction volumes may
`
`comprise, on average, greater than one copy of targeting oligonucleotid, and/or, on average, greater than one copy
`
`of target polynucleotide. Said reaction volumes may further comprise primers to a reference polynucleotide. In
`
`some cases, said reaction volumes further comprise a ligation probe to a reference polynucleotide.
`
`[0025]
`
`In some embodiments, the ligation probes are amplified within said reaction volumes.
`
`[0026] The fetal genetic material used in the methods for detecting a fetal genetic condition may be derived from a
`
`cellular sample that was selectively pre-enriched for fetal genetic material. But, in some embodiments, fetal genetic
`
`material used in the methods for detecting a fetal genetic condition is not derived from a cellular sample that was
`
`selectively pre—enriched for fetal genetic material
`
`[0027]
`
`The target polynucleotide may be within a chromosome selected from the group consisting of
`
`chromosome 18, 13, 21, and X; or from the group consisting of chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13,
`
`14,15,16,17,18,19,20, 21, 22, X, or Y.
`
`[0028]
`
`In some cases, said reaction volumes are aqueous droplets within an oil phase, said targeting
`
`oligonucleotides are ligation probes, and said determining step comprises comparing a number of droplets
`
`comprising an amplified product of said ligation probes with a number of droplets comprising an amplified product
`
`of ligation probes directed to a reference polynucleotide. In some cases, said reference polynucleotide is a region of
`
`a chromosome that is not associated with a fetal genetic abnormality.
`
`[0029] As used in the methods and compositions provided herein, said targeting oligonucleotides may be ligation
`
`probes that become circular upon ligation following hybridization to a target polynucleotide.
`
`[0030] This disclosure also provides compositions, such as microcapsule compositions, as well as methods for
`
`using said microcapsule compositions. In some cases, the composition is a microcapsule comprising a ligated probe
`
`wherein said microcapsule is obtained by: a.
`
`selectively binding a plurality of ligation probes to target
`
`polynucleotides within a genetic sample; b.
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`ligating a 5’ end of at least one of said bound ligation probes to a 3’
`
`end of the same or different bound ligation probe, thereby obtaining at least one ligation product; c.
`
`introducing an
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`aqueous solution comprising said at least one ligation product into a device for generating droplets; d. using said
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`device to produce an aqueous droplet comprising said at least one ligation product, wherein said aqueous droplet is
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`within an immiscible fluid; and
`
`e.
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`converting said droplet into a microcapsule comprising a solid-phase exterior. In some cases, said converting
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`comprises heating above 50 ° C, or heating above 70 ° C. The immiscible liquid (e.g., oil) may comprise a
`
`fluorinated surfactant. In some cases, the aqueous phase comprises a fluorinated surfactant. The oil may be a
`
`fluorocarbon oil. The oil phase may comprise an anionic surfactant. The oil phase may comprises ammonium
`
`Krytox. In some cases, said microcapsule does not comprise a bead bound to an oligonucleotide. Said
`
`microcapsule may remain substantially intact at temperatures above 70° C. The microcapsule may comprise ligation
`
`probes capable of selectively binding to a target polynucleotide associated with a genetic disorder. In some cases,
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`said ligation probes are capable of selectively binding to a target polynucleotide associated with fetal aneuploidy.
`
`In some cases, said genetic target is within a chromosome selected from the group consisting of chromosome 21,
`
`chromosome 13, chromosome 18, and the X chromosome. In some cases, the genetic target is within chromosome
`
`1, 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,17,18,19,20, 21, 22, X, or Y.
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`[0031] The microcapsule may comprise one or more of said ligation probes (e. g., padlock probe, molecular
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`inversion probe, ligation detection reaction (LDR) probe, circular probe, etc.). The microcapsules may comprise a
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`linear probe obtained by linearizing a probe previously circularized after a ligation reaction.
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`In some cases, the
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`microcapsules comprise circularized ligation probe. In some cases, the microcapsules contain linear products of a
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`ligation detection reaction (LDR).
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`[0032] The compositions and method provided herein may also relate to a water-in-oil mixture comprising two or
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`more aqueous droplets, wherein at least one of said two or more aqueous droplets comprises a first ligation probe
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`directed to a first target polynucleotide and at least one of said two or more aqueous droplets comprises a second
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`ligation probe directed to a second target polynucleotide. Said first target polynucleotide and said second target
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`polynucleotide may be the same molecule, or different molecules with identical sequences or structures, or different
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`molecules with different sequences or structures. In some cases, said first target polynucleotide has a different
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`sequence than that of said second target polynucleotide. In some instances, said first target polynucleotide has an
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`identical sequence to said second target polynucleotide. Said first target polynucleotide may comprise a first region
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`within a genomic segment and said second target polynucleotide may comprise a second region within said
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`genomic segment, wherein said first region does not have the same sequence as said second region.
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`[0033]
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`In some cases, said water-in-oil mixture further comprises an ammonium krytox surfactant. Said krytox
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`surfactant may be present in the oil phase of said mixture at a concentration of at least 0.01%.
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`[0034]
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`In some cases, said mixture comprises a ligation probe that is the linearized product of a circular probe
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`that was subjected to enzymatic cleavage. In some cases, said mixture comprises the circularized probe itself. In
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`some cases, the ligation probe may comprise an enzymatic cleavage site, such as where enzymatic cleavage is
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`catalyzed by uracil-N—glycosylase or a restriction enzyme.
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`[0035] The present invention includes a method of differential detection of target sequences in a mixture of
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`maternal and fetal genetic material, comprising the steps of: a) obtaining maternal tissue containing both maternal
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`and fetal genetic material; b) distributing the genetic material into discrete samples, each sample containing on
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`average not more than about one target sequence per sample, wherein the discrete sample contains a set of primers
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`to a known target sequence and/or a set of reference primers to a known reference sequence; c) performing an
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`amplification reaction; d) detecting the presence of the target or reference sequence in the discrete samples; and e)
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`comparing the ratio of target sequences detected to reference sequences detected to determine a differential amount
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`of target sequence. Said method may further comprise a step of comparing the ratio of target sequences detected to
`
`reference sequences detected to determine a differential amount of target sequence, wherein a difference in target
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`sequences detected to reference sequences detected indicates a fetal genetic abnormality.
`
`In some embodiments the
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`method is a method of detecting fetal aneuploidy. In some cases, the target sequence is a marker for aneuploidy and
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`the reference sequence is diploid in maternal and fetal genetic material. The maternal tissue may be maternal
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`peripheral blood, blood plasma or serum, or other tissue described herein. In some embodiments, the reaction
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`samples are in aqueous phases in an emulsion. In some cases, detecting the presence of the target or reference
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`sequence further includes hybridizing it in situ with a nucleic acid having a fluorescent label. In some cases, the
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`number of reaction samples is at least about 10,000. In some cases, steps b) to e) are repeated with a primer set to a
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`different target sequence. In some cases, the reaction volume comprises more than one primer set with each primer
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`set to a particular target sequence. In some cases, the reaction volume comprises more than one reference primer set
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`with each primer set to a particular reference sequence. Examples of primer sets that can be used include primer
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`sets specific for human chromosome 21, human chromosome 18, human chromosome 13, or human chromosome
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`X.
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`In some cases, aneuploidy is detected where the ratio of target to reference sequence detected is greater than 1.
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`In some cases, an aneuploidy is detected where the ratio of target to reference sequence detected is less than 1. In
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`some cases, the target sequence is at least a portion of a CFTR, Factor VIII (F8 gene), beta globin,
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`hemachromatosis, G6PD, neuroflbromatosis, GAPDH, beta amyloid, or pyruvate kinase gene.
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`INCORPORATION BY REFERENCE
`
`[0036] All publications and patent applications mentioned in this specification are herein incorporated by
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`reference in their entirety and to the same extent as if each individual publication or patent application was
`
`specifically and individually indicated to be incorporated by reference.
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`BRIEF DESCRIPTION OF THE FIGURES
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`[0037] The novel features of the invention are set forth with particularity in the appended claims. A better
`
`understanding of the features and advantages of the present invention will be obtained by reference to the following
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`detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized,
`
`and the accompanying figures of which:
`
`[0038] FIG. 1 is a schematic overview illustrating the steps that can be taken to detect copy number variations in a
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`patient sample through the use of droplet digital PCR and ligation probes.
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`[0039]
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`FIG. 2 is a schematic illustration of an example of steps that can be followed to detect changes in the
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`number of chromosomes (or portions thereof) in a sample.
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`[0040] FIG. 3 depicts a workflow of an exemplary method for diagnosing fetal aneuploidy.
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`[0041] FIG. 4 is a schematic illustration of the use of Molecular Inversion Probes (MIPS) to detect two genetic
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`targets.
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`[0042] FIG. 5 shows multiplexing of the MIP approach to increase sensitivity of detection of genetic targets.
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`[0043] FIG. 6 shows a two-color system for detection of nucleic acids in droplets using universal primers and
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`universal probes without cleavage.
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`[0044] FIG. 7 shows a scheme for detecting two genetic targets with two colors using a ligation-detection reaction
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`(LDR) followed by PCR in droplets.
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`[0045] FIG. 8 depicts the use of multiplexed oligonucleotides for LDR—PCR in droplets to enhance sensitivity of
`detection.
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`[0046] FIG. 9 depicts a computer useful for displaying, storing, retrieving, or calculating data or results obtained
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`by the methods and compositions described herein.
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`[0047] FIG. 10 shows a correlation