Noninvasive prenatal assay for autosomal recessive diseases

Abstract

Described in certain example embodiments herein are non-invasive prenatal assays, kits, and compositions for detection of autosomal recessive variant alleles.

Description

NONINVASIVE PRENATAL ASSAY FOR AUTOSOMAL RECESSIVE DISEASESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 589,871, filed on October 12, 2023, entitled “Noninvasive Prenatal Assay for Autosomal Recessive Diseases,” the contents of which is incorporated by reference herein in its entirety.SEQUENCE LISTING

[0002] This application contains a sequence listing filed in electronic form as an xml file entitled “SHOMOO-00001-U-PCT-01_ST26.xml”, created on October 8, 2024, and having a size of 540,501 bytes. The content of the sequence listing is incorporated herein in its entirety.TECHNICAL FIELD

[0003] The subject matter disclosed herein is generally directed to noninvasive prenatal assays.BACKGROUND

[0004] The search for adequate, safe prenatal diagnostics tools has been an ongoing quest for many years. Although amniocentesis and chorionic villi sampling (CVS) are still considered the gold standard for diagnostic prenatal testing, these procedures require a tertiary care setting at minimum and carry the risk of miscarriage. Thus, there exists a need for safer, inexpensive, noninvasive sampling techniques for diagnostic prenatal testing.

[0005] Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present invention.SUMMARY

[0006] Described in certain example embodiments herein is a method of detecting autosomal recessive variant alleles and / or autosomal recessive disorders in a fetus, the method including sequencing one or more regions of maternal genomic DNA (gDNA) comprising one or more target genes or one or more target gene variants, or both, of a mother of the fetus to determine a maternalgenotype variant allele frequency (VAF); sequencing one or more regions of circulating fetal DNA (cfDNA) comprising the one or more target genes in a biological sample obtained from the mother to determine a fetal genotype VAF; and detecting an allele imbalance from a maternal genotype VAF and the fetal genotype VAF thereby determining a genotype and a presence or absence of an autosomal recessive variant allele and / or disorder in the fetus.

[0007] In certain example embodiments, detecting an allele imbalance comprises determining a copy number of the maternal genotype VAF and the fetal genotype VAF.

[0008] In certain example embodiments, determining an allele imbalance includes performing a Kernel density estimation (KDE) analysis that comprises comparing a probability of the genotype of the maternal genotype VAF, fetal genotype VAF, or both to groups corresponding to a homozygous genotype, a heterozygous genotype, and a wild type genotype prepared by Kernel Density Estimation (KDE) analysis of a reference database for each of the one or more target genes.

[0009] In certain example embodiments, sequencing includes next generation sequencing. In certain example embodiments, sequencing is multiplexed sequencing. In certain example embodiments, includes generating one or more amplicons via polymerase chain reaction and sequencing the one or more amplicons.

[0010] In certain example embodiments, the method further includes performing methylation sensitive digestion of the maternal gDNA, cfDNA, or both prior to sequencing, generating one or more amplicons, or both.

[0011] In certain example embodiments, the one or more target genes are selected the group of: SMN1, ACADVL, ADA2, ADAT3, AGL, AK2, ALDH7A1, ARSA, ASL, B3GALT6, C12orf57, Ca2, CANT1, CEP290, CTFR, CNPY3, COG6, COL6A2, CUP2U1, DLD, EPCAM, FARS2, FKRP, GAA, GALC, GALNS, GBA, GLB1, GNPAT, GNPTG, GRM7, GUSB, HBB, HEXB, HIBCH, HSPG2, INVS, ISCA2, KIAA0586, LAMA1, LAMA2, LARGE1, LEPRE1, LIFR, MAN1B1, METTL5, MMUT, MUT, NDUFS4, NKX6-2, NPC1, NPHP3, NRAP, OTC, PCCA, PGAP3, PI4KA, PKHD1, PLA2G6, POLR1C, RAB3GAP1, RAG1, RAG2, RNASEH2C, SCO2, SUOX, SURF1, TBCE, TTN, WDR62, WNT7A.

[0012] In certain example embodiments, the one or more target gene variants are selected from the group consisting of: ACADVL c.65OA,p.Ser22Ter; ACADVL C.134OA, p.Ser45*;ACADVL c, 1048G>A, p.Gly35OArg / C.134OA, p.Ser45*; ACADVL C.134OA, p.Ser45*; ADA2 c.882-2A>G; ADAT3 c.430G>A,p.Vall44Met; AGL c.294-2A>T; AK2 c.524G>C,p.Argl75Pro; ALDH7A1 c.518-2A>C; ARSA c. H08-2A>G; ASL c,1060C<T, p.Gln354*; B3GALT6 c.77T>C, p.Leu26Pro; C12orf57 c.lA>G,p.MetlVal; Ca2 c.232+lG>A; CANT1 c. 894_898dupGCCGC, p.Ser303Alafs*21; CANT1 c.902_906dupGCGCC, р.Ser303fs*21; CFTR c,1418del; CFTR p.Gly473Glufs*54; CNPY3 c.496-3_496-2delCA; C0G6 с, 1167-24A>G; COL6A2 c.2422+lG>A; COL6A2 C.1817-1G>A; CYP2U1 c.947A>T,p.Asp316Val; DLD c.685G>T,p.Gly229Cys; EPCAM c.583dup; EPCAM c.499dup, р.Glnl67Profs*21; EPCAM c.367G>C,p.Glyl23Arg; EPCAM c.451G>C; FARS2 с.431A>G,p.Tyrl44Cys; FKRP c.941C>T, p.Thr314Met; FKRP c, 1364C>A, p.Ala455Asp; GAA c. l657C>T,p.Q553*; GAA c, 1657C>T,p.Q553X; GALC c, 1886G>T,p.W629L; GALNS c.958G>A, p.Glu320Lys; GALNS c.970G>C, p.Ala324Pro; GBA c, 1448T>C,p.Leu483Pro; GLB1 c.622C>T,p.Arg208Cys; GNPAT c.569-3T>G; GRM7 c.461T>C ,p.Ilel54Thr; GUSB c. l832G>A,p.Arg611Gln; HBB c.315+lG>A; HEXB c.94C>T, p.Gln32*; HIBCH c.410C>T, p.Alal37Val; HSPG2 c.l 1674+1G>T; INVS c,1807C>T, p.Arg603*; ISCA2 c.229G>A, р.Gly77Ser; KIAA0586 c.4191del , p.Argl397fs; LAMA1 c.6779C>A, p.Ser2260Tyr; LAMA2 с.3924+2T>C; LAMA2 c,1762del,p.Ala588Leufs*l 1; LARGE1 exon 5-7 deletion; LEPRE1 c, 1839-2A>C; LIFR c.l258C>T, p.Gln420Ter; MAN1B1 c,1376del,p.Gly459Alafs*12; METTL5 c.541+lG>C; MMUT c.810_81 ldelins,p.Ala271Leufs*l 1; MUT c.785G>T,p.Ser262Ile; NDUFS4 c.350+lG>A; NKX6-2 c,196delC,p.R66fs*122; NPC1 c.2130+lG>A; NPC1 c.3609_3610delAC, p.L1204YfsX53; NPHP3 c.3675dupT,p.Alal226fs; NPHP3 c.3674_3675insA; NRAP c.400_407del, p.Cysl34Serfs*12; OTC c.275G>A; p.Arg92Gln; PCCA c.l l l_130del,p.Tyr391Asnfs*5; PCCA c. 425G>A,p.Glyl42Asp; PGAP3 c.850C>T; р.His284Tyr; PI4KA c.3O53C>T ,p.SerlO18Leu; PKHD1 c.9374C>G, p.Ser3125*; PKHD1 с.2180A>G,p.Asn727Ser; PLA2G6 c.2070_2072del; p.Val691del; PLA2G6 c, 1933C>T; р.Arg645*; POLR1C c.934T>C, p.Ser312Pro; RAB3GAP1 c,1009C>T,p.Arg337*; RAG1 с.555del,p.Lysl86Serfs*15; RAG2 c, 1332C>G, p.Ile444Met; RNASEH2C c.202C>G,p.Leu68Val; SCO2 c.2T>C / pMetl; SMN1 deletion of exons 7 and 8; SUOX c.520del; p.Aspl74Thrfs*13; SURF1 c.758_759del,p.Thr253Serfs*38; TBCE c,155_166dell2bp,p.S52_G55del; TTN c.35054del,p.Hisl l685Leufs*15; WDR62 c.758_760delACA, p.Asn253del; and WNTA c.874C>T,p.Arg292Cys.

[0013] In certain example embodiments, the method further includes sequencing one or more positive control spike-in fragmented DNA samples each corresponding to a 0, 1, or 2 copy number of the one or more target genes.

[0014] In certain example embodiments, the maternal genomic DNA, the cfDNA, or both are from blood or component thereof of the mother. In certain example embodiments, the blood or component thereof is plasma.

[0015] Described in certain example embodiments, herein are methods of treating or preventing an autosomal recessive disorder or a symptom thereof in a fetus including detecting a presence of an autosomal recessive disorder in the fetus by performing a method of the present description; and treating or preventing the autosomal recessive disorder or a symptom thereof during gestation or post-partum.

[0016] In certain example embodiments, treating or preventing comprises administering a genetic modifying agent to the fetus during gestation and / or to a neonate, child, or adult postpartum, where the genetic modifying agent is configured to modify the variant allele causative of the autosomal recessive disease or disorder such that it is an allele variant that does not result in autosomal recessive disease or disorder.

[0017] Described in certain example embodiments herein is a kit including one or more primer sets, wherein each primer set is configured to amplify a region of a target gene of an autosomal recessive variant allele and / or autosomal recessive disorder and are further configured for use in a method of the present description.

[0018] In certain example embodiments, the target gene is selected from the group consisting of: SMN1, ACADVL, ADA2, ADAT3, AGL, AK2, ALDH7A1, ARSA, ASL, B3GALT6, C12orf57, Ca2, CANT1, CEP290, CTFR, CNPY3, COG6, COL6A2, CUP2U1, DLD, EPCAM, FARS2, FKRP, GAA, GALC, GALNS, GBA, GLB1, GNPAT, GNPTG, GRM7, GUSB, HBB, HEXB, HIBCH, HSPG2, INVS, ISCA2, KIAA0586, LAMA1, LAMA2, LARGE1, LEPRE1, LIFR, MAN1B1, METTL5, MMUT, MUT, NDUFS4, NKX6-2, NPC1, NPHP3, NRAP, OTC, PCCA, PGAP3, PI4KA, PKHD1, PLA2G6, POLR1C, RAB3GAP1, RAG1, RAG2, RNASEH2C, SCO2, SUOX, SURF1, TBCE, TTN, WDR62, WNT7A, or any combination thereof.

[0019] In certain example embodiments, the target gene is a target gene variant selected from the group consisting of: ACADVL c.65C>A,p.Ser22Ter; ACADVL C.134OA, p.Ser45*; ACADVL c. 1048G>A, p.Gly35OArg / C.134OA, p.Ser45*; ACADVL c, 134C>A, p.Ser45*; ADA2 c.882-2A>G; ADAT3 c.430G>A,p.Vall44Met; AGL c.294-2A>T; AK2 c.524G>C,p.Argl75Pro; ALDH7A1 c.518-2A>C; ARSA c,1108-2A>G; ASL c,1060C<T, p.Gln354*; B3GALT6 c.77T>C, p.Leu26Pro; C12orf57 c.lA>G,p.Metl Vai; Ca2 c.232+lG>A; CANT1 c. 894_898dupGCCGC, p.Ser303Alafs*21; CANT1 c.902_906dupGCGCC, р.Ser303fs*21; CFTRc.l418del; CFTR p.Gly473Glufs*54; CNPY3 c.496-3_496-2delCA; COG6 с, 1167-24A>G; COL6A2 c.2422+lG>A; COL6A2 C.1817-1G>A; CYP2U1 c.947A>T,p.Asp316Val; DLD c.685G>T,p.Gly229Cys; EPCAM c.583dup; EPCAM c.499dup, р.Glnl67Profs*21; EPCAM c.367G>C,p.Glyl23Arg; EPCAM c.451G>C; FARS2 с.431A>G,p.Tyrl44Cys; FKRP C.941OT, p.Thr314Met; FKRP c, 1364C>A, p.Ala455Asp; GAA c. l657C>T,p.Q553*; GAA c, 1657C>T,p.Q553X; GALC c,1886G>T,p.W629L; GALNS c.958G>A, p.Glu320Lys; GALNS c.970G>C, p.Ala324Pro; GBA c. 1448T>C,p.Leu483Pro; GLB1 c.622C>T,p.Arg208Cys; GNPAT c.569-3T>G; GRM7 c.461T>C ,p.Ilel54Thr; GUSB c. l832G>A,p.Arg611Gln; HBB c.315+lG>A; HEXB c.94C>T, p.Gln32*; HIBCH c.410C>T, p.Alal37Val; HSPG2 c.l 1674+1G>T; INVS c,1807C>T, p.Arg603*; ISCA2 c.229G>A, р.Gly77Ser; KIAA0586 c.4191del , p.Argl397fs; LAMA1 c.6779C>A, p.Ser2260Tyr; LAMA2 с.3924+2T>C; LAMA2 c,1762del,p.Ala588Leufs*l 1; LARGE1 exon 5-7 deletion; LEPRE1 c.1839-2A>C; LIFR c.1258C>T, p.Gln420Ter; MAN1B 1 c.1376del,p.Gly459Alafs* 12; METTL5 c.541+lG>C; MMUT c.810_811delins,p.Ala271Leufs*l l; MUT c.785G>T,p.Ser262Ile; NDUFS4 c.350+lG>A; NKX6-2 c,196delC,p.R66fs*122; NPC1 c.2130+lG>A; NPC1 c.3609_3610delAC, p.L1204YfsX53; NPHP3 c.3675dupT,p.Alal226fs; NPHP3 c.3674_3675insA; NRAP c.400_407del, p.Cysl34Serfs*12; OTC c.275G>A; p.Arg92Gln; PCCA c.l l l_130del,p.Tyr391Asnfs*5; PCCA c. 425G>A,p.Glyl42Asp; PGAP3 c.850C>T; р.His284Tyr; PI4KA c.3O53C>T ,p.Serl018Leu; PKHD1 c.9374C>G, p.Ser3125*; PKHD1 с.2180A>G,p.Asn727Ser; PLA2G6 c.2070_2072del; p.Val691del; PLA2G6 c, 1933C>T; р.Arg645*; POLR1C c.934T>C, p.Ser312Pro; RAB3GAP1 c,1009C>T,p.Arg337*; RAG1 с.555del,p.Lysl86Serfs*15; RAG2 c, 1332C>G, p.Ile444Met; RNASEH2C c.202C>G,p.Leu68Val; SC02 c.2T>C / p.Metl; SMN1 deletion of exons 7 and 8; SUOX c.520del;p.Aspl74Thrfs*13; SURF1 c.758_759del,p.Thr253Serfs*38; TBCE c,155_166dell2bp, p.S52_G55del; TTN c.35054del,p.Hisl l685Leufs*15; WDR62 c.758_760delACA, p.Asn253del; and WNTA c.874OT,p.Arg292Cys, or any combination thereof.

[0020] In certain example embodiments, the kit further includes one or more reagents for carrying out polymerase chain reaction, sequencing, or both.

[0021] These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of example embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention may be utilized, and the accompanying drawings of which:

[0023] FIG. 1 shows a graph demonstrating that three (3) groups of normalized data points by PAF are distinctly populated.

[0024] FIG. 2A-2B show graphs demonstrating AVAF normalized by NGS PAF for plasma cfDNA (FIG. 2A) and fetal HOM, HET, and WT (FIG. 2B).

[0025] FIG. 3 shows a graph demonstrating the correlation of ff and spik-in levels.

[0026] FIG. 4 shows a graph demonstrating the correlation of calculated mixture copy numbers and expected copy number.

[0027] FIG. 5 shows a graph demonstrating calculated fetal DNA copy number.

[0028] FIG. 6 shows a graph demonstrating results from a KDE analysis for determining fetal genotype.

[0029] FIG. 7 shows a graph demonstrating results from a KDE analysis for determining fetal genotype with results considered as “low confidence” identified at “A” and those results considered “high confidence” identified at “B”.

[0030] FIG. 8 shows an experimental design for fetal fraction determination. Methylation nonsensitive digestion is used as a negative control.

[0031] FIG. 9 shows a flow diagram of processing of the sequencing data.

[0032] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS

[0033] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0035] All publications and patents cited in this specification are cited to disclose and describe the methods and / or materials in connection with which the publications are cited. All such publications and patents are herein incorporated by references as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Such incorporation by reference is expressly limited to the methods and / or materials described in the cited publications and patents and does not extend to any lexicographical definitions from the cited publications and patents. Any lexicographical definition in the publications and patents cited that is not also expressly repeated in the instant application should not be treated as such and should not be read as defining any terms appearing in the accompanying claims. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0036] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recitedmethod can be carried out in the order of events recited or in any other order that is logically possible.

[0037] Where a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of Tess than x’, less than y’, and Tess than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0038] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0039] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or subranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.General Definitions

[0040] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2ndedition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4thedition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR2: A Practical Approach (1995) (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2ndedition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlett, 2008 (ISBN 0763752223); Kendrew e / czZ. (eds ), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2ndedition (2011).

[0041] Definitions of common terms and techniques in chemistry and organic chemistry can be found in Smith. Organic Synthesis, published by Academic Press. 2016; Tinoco et al. PhysicalChemistry, 5thedition (2013) published by Pearson; Brown et al., Chemistry, The Central Science 14thed. (2017), published by Pearson, Clayden et al., Organic Chemistry, 2nded. 2012, published by Oxford University Press; Carey and Sunberg, Advanced Organic Chemistry, Part A: Structure and Mechanisms, 5thed. 2008, published by Springer; Carey and Sunberg, Advanced Organic Chemistry, Part B: Reactions and Synthesis, 5thed. 2010, published by Springer, and Vollhardt and Schore, Organic Chemistry, Structure and Function; 8thed. (2018) published by W.H. Freeman.

[0042] Definitions of common terms, analysis, and techniques in genetics can be found in e.g., Hartl and Clark. Principles of Population Genetics. 4thEd. 2006, published by Oxford University Press. Published by Booker. Genetics: Analysis and Principles, 7thEd. 2021, published by McGraw Hill; Isik et la., Genetic Data Analysis for Plant and Animal Breeding. First ed. 2017. published by Springer International Publishing AG; Green, E. L. Genetics and Probability in Animal Breeding Experiments. 2014, published by Palgrave; Bourdon, R. M. Understanding Animal Breeding. 2000 2ndEd. published by Prentice Hall; Pal and Chakravarty. Genetics and Breeding for Disease Resistance of Livestock. First Ed. 2019, published by Academic Press; Fasso, D. Classification of Genetic Variance in Animals. First Ed. 2015, published by Callisto Reference; Megahed, M. Handbook of Animal Breeding and Genetics, 2013, published by Omniscriptum Gmbh & Co. Kg., LAP Lambert Academic Publishing; Reece. Analysis of Genes and Genomes. 2004, published by John Wiley & Sons. Inc; Deonier et al., Computational Genome Analysis. 5thEd. 2005, published by Springer- Verlag, New York; Meneely, P. Genetic Analysis: Genes, Genomes, and Networks in Eukaryotes. 3rdEd. 2020, published by Oxford University Press.

[0043] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0044] As used herein, "about," "approximately," “substantially,” and the like, when used in connection with a measurable variable such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value including those within experimental error (which can be determined by e.g. given data set, art accepted standard, and / or with e.g., a given confidence interval (e.g. 90%, 95%, or more confidence interval from the mean), such as variations of + / -10% or less, +7-5% or less, + / -!% or less, and + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention.As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” can mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0045] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0046] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0047] As used herein, a “biological sample” refers to a sample obtained from, made by, secreted by, excreted by, or otherwise containing part of or from a biologic entity. A biologic sample can contain whole cells and / or live cells and / or cell debris, and / or cell products, and / or virus particles. The biological sample can contain (or be derived from) a “bodily fluid”. The biological sample can be obtained from an environment (e.g., water source, soil, air, and the like). Such samples are also referred to herein as environmental samples. As used herein “bodily fluid” refers to any non-solid excretion, secretion, or other fluid present in an organism and includes, without limitation unless otherwise specified or is apparent from the description herein, amniotic fluid, aqueous humor, vitreous humor, bile, blood or component thereof (e.g. plasma, serum, etc.), breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixturesof one or more thereof. Biological samples include cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids may be obtained from an organism, for example by puncture, or other collecting or sampling procedures.

[0048] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0049] As used herein, “administering” refers to any suitable administration for the agent(s) being delivered and / or subject receiving said agent(s) and can be oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intraosseous, intraocular, intracranial, intraperitoneal, intralesional, intranasal, intracardiac, intraarticular, intracavernous, intrathecal, intravireal, intracerebral, and intracerebroventricular, intratympanic, intracochlear, rectal, vaginal, by inhalation, by catheters, stents or via an implanted reservoir or other device that administers, either actively or passively (e.g. by diffusion) a composition the perivascular space and adventitia. For example, a medical device such as a stent can contain a composition or formulation disposed on its surface, which can then dissolve or be otherwise distributed to the surrounding tissue and cells. The term “parenteral” can include subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intrastemal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques. Administration routes can be, for instance, auricular (otic), buccal, conjunctival, cutaneous, dental, electro-osmosis, endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intraarterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracisternal, intracorneal, intracoronal (dental), intracoronary, intracorporus cavemosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous,intravenous bolus, intravenous drip, intraventricular, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric, occlusive dressing technique, ophthalmic, oral, oropharyngeal, other, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, and / or vaginal administration, and / or any combination of the above administration routes, which typically depends on the disease to be treated, subject being treated, and / or agent(s) being administered.

[0050] The term “blood”, as used herein, refers whole blood. This can include whole blood with or without additives, diluents, or other compositions, non-blood cells, etc. added to it. Blood includes blood obtained from a single source and pools of blood from multiple sources. The term “blood product” refers to any therapeutic product produced from or with blood or a component thereof. The term “blood component” can refer to any part, fraction, cell, or molecule of blood. Often the phrase “blood, blood product, and components thereof’ is used herein. This is to mean blood, blood products, blood components, and blood product components. In some embodiments, blood components are also blood products and vice versa. In some embodiments, blood is also a blood product. Also, derivatives of blood components, such as plasma derivatives, are considered blood products as the term is used herein. “Blood components” can include, but is not limited to, red blood cells, white blood cells, platelets, serum, and plasma.

[0051] As used herein, the terms “disease” or “disorder” are used interchangeably throughout this specification, and refer to any alternation in state of the body or of some of the cells and / or cellular processes, tissues, and / or organs, interrupting or disturbing the performance of the functions thereof and / or causing symptoms such as discomfort, dysfunction, distress, cognitive impairment, illness, or even death to the person afflicted or those in contact with a person. A disease or disorder can also be related to a distemper, ailing, ailment, malady, disorder, sickness, illness, complaint, indisposition, or affliction.

[0052] As used herein, “gene” refers to a hereditary unit corresponding to a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a characteristic(s) or trait(s) in an organism. The term gene can refer to translated and / or untranslated regions of a genome. “Gene” can refer to the specific sequence of DNA that is transcribed into anRNA transcript that can be translated into a polypeptide or be a catalytic RNA molecule, including but not limited to, tRNA, siRNA, piRNA, miRNA, long-non-coding RNA and shRNA.

[0053] As used herein, “isolated” means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature. A non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, do not require “isolation” to distinguish it from its naturally occurring counterpart.

[0054] As used herein, “nucleic acid,” “nucleotide sequence,” and “polynucleotide” can be used interchangeably herein and can generally refer to a string of at least two base-sugar-phosphate combinations and refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, polynucleotide as used herein can refer to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions can be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triplehelical region often is an oligonucleotide. “Polynucleotide” and “nucleic acids” also encompasses such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. For instance, the term polynucleotide as used herein can include DNAs or RNAs as described herein that contain one or more modified bases. Thus, DNAs or RNAs including unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. “Polynucleotide”, “nucleotide sequences” and “nucleic acids” also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids. Natural nucleic acids have a phosphate backbone, artificial nucleic acids can contain other types of backbones, but contain the same bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “nucleic acids” or "polynucleotides" as that term is intended herein. As used herein, “nucleic acidsequence” and “oligonucleotide” also encompasses a nucleic acid and polynucleotide as defined elsewhere herein.

[0055] As used herein, “preventative” and “prevent” refers to hindering or stopping a disease or condition before it occurs, even if undiagnosed, or while the disease or condition is still in the sub-clinical phase.

[0056] As used herein, “therapeutic” refers to treating, healing, and / or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect.

[0057] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0058] As used herein, the terms "treating" and "treatment" can refer generally to obtaining a desired pharmacological and / or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof, such as an autosomal recessive disease or disorder, such as that caused by an autosomal recessive variant allele. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term "treatment" as used herein covers any treatment of an autosomal recessivedisease or disorder, such as that caused by an autosomal recessive variant allele, in a subject, particularly a human or human fetus, and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and / or its symptoms or conditions. The term "treatment" as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with the disorder and / or those in which the disorder is to be prevented. As used herein, the term "treating", can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and / or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

[0059] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.OVERVIEW

[0060] Autosomal recessive diseases (ARDs), a subset of genetic disorders responsible for considerable disease burden, affect approximately 1.7-5 in 1000 neonates (Xiao Q, Lauschke VM. The prevalence, genetic complexity and population-specific founder effects of human autosomal recessive disorders. NPJ Genom Med. 2021 ;6(1):41. Epub 20210602). ARDs have a higher prevalence in populations with high rates of consanguinity (Xiao Q, Lauschke VM. The prevalence, genetic complexity and population-specific founder effects of human autosomal recessive disorders. NPJ Genom Med. 2021;6(l):41. Epub 20210602). Over a billion people globally live in communities where consanguineous marriage is a traditional and respected social trend of marital union (Bener et al. Egyptian Journal of Medical Human Genetics. 2017; 18(4):315- 20). Specific regions with a high ratio of consanguineous marriage are the Middle East, South andWest Asia, and sub-Saharan African societies (Bener et al. Egyptian Journal of Medical Human Genetics. 2017;18(4):315-20). For example, Tay-Sachs disease is about 100 times more frequent in Ashkenazi Jews compared to other populations (Xiao Q, Lauschke VM. NPJ Genom Med. 2021 ;6(1):41. Epub 20210602). Sickle cell disease occurs in approximately 1 out of every 365 births of African- American infants in the US, and about 1 in 13 African American babies in the US are born with the sickle cell trait (Centers for Disease Control and Prevention. Data & Statistics on Sickle Cell Disease Washington, DC: U.S. Department of Health & Human Services; 2022 [cited 2023 March 24], Available from: https: / / www.cdc.gov / ncbddd / sicklecell / data.html). In sub- Saharan populations, the incidence rate for sickle cell disease is 80x higher than in the US (Zhou et al., New England Journal of Medicine. 2022;387(6):488-91).

[0061] More than 2000 ARDs exist and symptoms of ARD usually begin early in life, but no cure exists for most ARDs. Therapies may be available to treat or manage some of the associated signs or symptoms, and a few disorders have been treated with gene therapy or enzyme replacement. Prenatal diagnostics and early intervention before symptoms begin are the best approach to prevent ARD. The gold standard for prenatal screening of ARDs continues to be amniocentesis and chorionic villous sampling (CVS); however, these procedures are invasive and carry the risk of pregnancy loss (Xiao Q, Lauschke VM. The prevalence, genetic complexity and population-specific founder effects of human autosomal recessive disorders. NPJ Genom Med. 2021;6(l):41. Epub 20210602). They are also expensive and must be done in a tertiary care setting.

[0062] The search for adequate, safe prenatal diagnostics tools has been an ongoing quest for many years. Although amniocentesis and chorionic villi sampling (CVS) are still considered the gold standard for diagnostic prenatal testing, these procedures require a tertiary care setting at minimum and carry the risk of miscarriage. The demand for safer, inexpensive, noninvasive sampling techniques has increased substantially in recent years. One such option that in clinical use is cell-free noninvasive prenatal testing or screening (NIPT / NIPS). The discovery of circulating cell-free DNA (cfDNA) in the plasma of the pregnant women provides the foundation to develop noninvasive prenatal testing (NIPT). NIPT is currently mainly used for the detection of common aneuploidies such as trisomy 13, 18 and 21, and has a higher sensitivity and specificity compared to the traditional maternal serum screening. Cell-free NIPT rapidly evolved to become one of the standard components of prenatal care, and it is estimated that already more than 6 millionwomen have undergone NIPT pregnancy screening. While clinically utilized NIPT succeeds in screening for chromosomal abnormalities and monogenic autosomal dominant disorders, NIPT for monogenic autosomal recessive disorders (ARD) remains a challenge for clinical application.

[0063] With that said, embodiments disclosed herein describe NIPT assays, compositions, and methods that can be capable of detecting autosomal recessive disorders and other characteristics. Other compositions, compounds, methods, features, and advantages of the present disclosure will be or become apparent to one having ordinary skill in the art upon examination of the following drawings, detailed description, and examples. It is intended that all such additional compositions, compounds, methods, features, and advantages be included within this description, and be within the scope of the present disclosure.DETECTING AUTOSOMAL RECESSIVE ALLELIC VARIANTS

[0064] Described herein are methods of detecting autosomal recessive allelic variants, such as autosomal recessive allelic variants associated with diseases, disorders, conditions, or other phenotypes, such as those not necessarily associated with a pathology, in a fetus. In certain example embodiments a method of detecting autosomal recessive allelic variants (e.g., autosomal recessive disorders) in a fetus includes sequencing one or more regions of maternal genomic DNA (gDNA) containing one or more target genes and / or variants of a mother of the fetus to determine a maternal genotype variant allele frequency (VAF); sequencing one or more regions of circulating fetal DNA (cfDNA) containing the one or more target genes and / or variants in a biological sample obtained from the mother to determine a fetal genotype VAF; detecting an allele imbalance from the maternal genotype VAF and the fetal genotype VAF thereby determining the genotype and the presence or absence of an autosomal recessive allelic variant and / or autosomal recessive disorder in the fetus. In certain example embodiments, detecting an allele imbalance includes determining copy number of the maternal genotype VAF and the fetal genotype VAF.

[0065] In certain example embodiments, determining an allele imbalance includes performing a Kernel density estimation (KDE) analysis that includes comparing a probability of the genotype of the maternal genotype VAF, fetal genotype VAF, or both to groups corresponding to the homozygous, heterozygous, and wild type genotypes prepared by KDE analysis of a reference database for each of the one or more target genes. In general KDE analyses involves the application of kernel smoothing for probability density estimation and has been used to identify the associationbetween genes and quantitative traits (see e.g., Yee et al., Identification of the associations between genes and quantitative traits using entropy-based kernel density estimation. Genomics Inform 2022, 20(2):el7; Wang et al., Brief Bioinform. 2015 Mar; 16(2): 183-192). The KDE analysis can allow determining the probability of a fetal genotype as wild-type (or not carrying the autosomal recessive allelic variant), homozygous for the autosomal recessive allelic variant, or heterozygous for the autosomal recessive allelic variant.Nucleic Acid Amplification and Sequencing

[0066] In some embodiments, the method of detecting autosomal recessive variants of the present disclosure include amplicon sequencing, which generally includes amplifying DNA, such as maternal genomic DNA and / or cfDNA (e.g., fetal cfDNA) present in a sample, by performing an amplification reaction to form amplicons. The amplicons are then sequenced by a suitable sequencing technique such as a next-generation sequencing method. In some embodiments, amplicon generation is not specific for a target gene or variant of interest. In some embodiments, primers for amplicon generation are designed such that they are tiled. In some embodiments amplicon generation is specific for a target gene or variant of interest. In some embodiments, both specific and non-specific amplification of DNA, such as maternal genomic DNA and / or cfDNA (e.g., fetal cfDNA) present in a sample is performed.

[0067] In certain example embodiments, the DNA amplification is an isothermal amplification (see e.g., Zhao et al., Chem Rev. 2015 Nov 25;115(22): 12491-545; Mayboroda et al., Anal Biochem. 2018 Mar 15;545:20-30; Yan et al., Mol Biosyst. 2014 May;10(5):970-1003). In certain example embodiments, the isothermal amplification may be nucleic-acid sequence-based amplification (NASBA) (see e.g., Malek et al., 1994. Methods in Molecular Biology. Vol. 28. pp. 253-260; Yan et al., Mol Biosyst. 2014 May;10(5):970-1003; Deiman et al., 2002. Molecular Biotechnology. 20 (2): 163-180), recombinase polymerase amplification (RPA) (see e.g., Yan et al., Mol Biosyst. 2014 May;10(5):970-1003; Lobato and O’Sullivan. Trends Analyt Chem. 2018 Jan;98: 19-35; and Tan et al., Front Cell Infect Microbiol. 2022 Nov 28; 12: 1019071), loop- mediated isothermal amplification (LAMP) (see e.g., Notomi et al., J Microbiol. 2015 Jan;53(l):l- 5; Soroka et al., Cells. 2021 Jul 29; 10(8): 1931. doi: 10.3390 / cellsl0081931; and Varona et al., ACS Omega. 2021 Jan 28;6(5):3463-3469), strand displacement amplification (SDA) (see e.g., Yan et al., Mol Biosyst. 2014 May;10(5):970-1003; Walker, G.T., PCR Methods Appl. 1993Aug;3(l): 1-6. doi: 10.1101 / gr.3.1.1.), helicase-dependent amplification (HD A), or a nickase-based amplification reaction (e.g., nicking enzyme amplification reaction (NEAR) or nicking enzyme assisted amplification (NEAA), or a Cas-based nickase amplification reaction) (see e.g., Yan et al., Mol Biosyst. 2014 May; 10(5):970-1003; Qian et al., Anal Chim Acta. 2019 Mar 7; 1050: 1-15. doi: 10.1016 / j.aca.2018.10.054; ). In certain example embodiments, non-isothermal amplification methods may be used which include, but are not limited to, PCR, multiple displacement amplification (MDA) (see e.g., Lasken, R.S., Biochem Soc Trans. 2009 Apr;37(Pt 2):450-3. doi: 10.1042 / BST0370450), rolling circle amplification (RCA) (see e.g., Ali et al., Chem Soc Rev. 2014 May 21;43(10):3324-41. doi: 10.1039 / c3cs60439j; Goo et al., Biochip J. 2016;10(4):262- 271. doi: 10.1007 / sl3206-016-0402-6), ligase chain reaction (LCR) and other ligase-based nucleic acid amplification methods (see e.g., Li et al., Mol Cell Biochem. 2023 Jul;478(7): 1621-1631), or ramification amplification method (RAM) (see e.g., Zhang et al., Mol Diagn. 2001 Jun;6(2):141- 50. doi: 10.1054 / modi.2001.25323). See also e g., Additional amplification methods suitable for use with the present disclosure will be appreciated by those of ordinary skill in the art in view of the present disclosure.

[0068] In some embodiments, amplification includes incubating the sample (or nucleic acids therein or extracted therefrom) or set of samples under conditions sufficient for an enzymatic reaction to occur. In certain example embodiments, the incubation time of the present invention may be shortened. One skilled in the art can perform biochemical reactions in 5 minutes (e.g., 5 minute ligation, extension, etc ). Incubating may occur at one or more temperatures over time frames between about 10 minutes and 3 hours, preferably less than 200 minutes, 150 minutes, 100 minutes, 75 minutes, 60 minutes, 45 minutes, 30 minutes, or 20 minutes, depending on sample, reagents and components of the system. In some embodiments, incubating is performed at one or more temperatures between about 20° C and 100° C, in certain embodiments or about 37° C, in some embodiments, between about 45 °C to 75° C, 55 to 70 °C or 60 °C to 65 °C. In some embodiments, incubating is performed at one or more temperatures (such as in one or more incubation steps) at a temperature that is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 °C. In some embodiments,incubation is performed in one or more steps where each step is performed at a temperature ranging from about 20, to about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 °C. In some embodiments, incubation is performed in one or more steps where each step is performed at a temperature ranging from about 45, to 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 °C. In some embodiments, incubation is performed in one or more steps where each step is performed at a temperature ranging from about 55 to 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, or 70 °C. In some embodiments, incubation is performed in one or more steps where each step is performed at a temperature ranging from about 60 to 61, 62, 63, 64, or 65 °C.

[0069] Primer and amplicon design considerations and tools for amplicon sequencing library generation are described herein (see e.g., Working Examples) and generally known in the art. See e.g., Quick et al., Nature Protocols volume 12, pages 1261-1276 (2017); Gervais et al., Nucleic Acids Res. 2010 Jul 1; 38(Web Server issue): W308-W312; Taylor et al., Front. Cell. Infect. Microbiol. 2020. 10: Article 565591; Gohl et al., BMC Genomics volume 21, Article number: 863 (2020); Wienzek-Lischka et al., Transfus Med Hemother. 2020 Feb;47(l):14-22. doi: 10.1159 / 000505161; Francis et al., Scientific Reports volume 7, Article number: 44437 (2017), which can be adapted for use with the present disclosure. Amplicons for sequencing can be generated using any suitable approach. In some embodiments, barcodes or other identifiers and / or sequencing adapters can be incorporated during amplification. Barcodes and sequencing adapters are described elsewhere herein and will be appreciated by one of ordinary skill in the art in view of the present disclosure. Several exemplary amplification techniques are described herein and others suitable for use with the methods herein will be appreciated by those of ordinary skill in the art in view of this disclosure.

[0070] The amplification methods and compositions can include the use of various amplification reagents. Different components or reagents useful for amplification of nucleic acids are described herein and others will be appreciated by those of ordinary skill in the art in view of the present disclosure. For example, an amplification reagent as described herein may include a buffer, such as a Tris buffer. A Tris buffer may be used at any concentration appropriate for thedesired application or use, for example including, but not limited to, a concentration of ImM to 1 M, such as about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, 800 mM, 825 mM, 850 mM, 875 mM, 900 mM, 925 mM, 950 mM, 975 mM, to / or 1 M, or the like. One of ordinary skill in the art will be able to determine an appropriate concentration of a buffer such as Tris for use with the present invention.

[0071] A salt, such as magnesium chloride (MgCL), potassium chloride (KC1), or sodium chloride (NaCl), can be included in an amplification reaction, such as PCR, in order to improve the amplification of nucleic acid fragments. Although the salt concentration will depend on the particular reaction and application, in some embodiments, nucleic acid fragments of a particular size may produce optimum results at particular salt concentrations. Larger products may require altered salt concentrations, typically lower salt, in order to produce desired results, while amplification of smaller products may produce better results at higher salt concentrations. One of skill in the art will understand that the presence and / or concentration of a salt, along with alteration of salt concentrations, may alter the stringency of a biological or chemical reaction, and therefore any salt may be used that provides the appropriate conditions for a reaction of the present disclosure and as described herein.

[0072] In some embodiments, amplification reagents as described herein may be appropriate for use in hot-start amplification. Hot start amplification may be beneficial in some embodiments to reduce or eliminate dimerization of adaptor molecules or oligos, or to otherwise prevent unwanted amplification products or artifacts and obtain optimum amplification of the desired product. Many components described herein for use in amplification may also be used in hot-start amplification. In some embodiments, reagents or components appropriate for use with hot-start amplification may be used in place of one or more of the composition components as appropriate. For example, a polymerase or other reagent may be used that exhibits a desired activity at a particular temperature or other reaction condition. In some embodiments, reagents may be used that are designed or optimized for use in hot-start amplification, for example, a polymerase may be activated after transposition or after reaching a particular temperature. Such polymerases maybe antibody-based or aptamer-based. Polymerases as described herein are known in the art. Examples of such reagents may include, but are not limited to, hot-start polymerases, hot-start dNTPs, and photo-caged dNTPs. Such reagents are known and available in the art. One of skill in the art will be able to determine the optimum temperatures as appropriate for individual reagents.

[0073] Amplification of nucleic acids may be performed using specific thermal cycle machinery or equipment, and may be performed in single reactions or in bulk, such that any desired number of reactions may be performed simultaneously. In some embodiments, amplification may be performed using microfluidic or robotic devices, or may be performed using manual alteration in temperatures to achieve the desired amplification. In some embodiments, optimization may be performed to obtain the optimum reactions conditions for the particular application or materials. One of skill in the art will understand and be able to optimize reaction conditions to obtain sufficient amplification.

[0074] In certain embodiments, detection of DNA with the methods or systems of the disclosure requires transcription of the (amplified) DNA into RNA prior to detection.

[0075] Additional amplification regents are provided herein, see e.g., “Kits” that can be used with methods and compositions of the present disclosure.Sequencing

[0076] After amplification of amplicons, the amplicons can be sequenced via a next generation sequencing (NGS) technique or other suitable sequencing technique. Exemplary NGS techniques are described herein and others will be appreciated by one of ordinary skill in the art in view of the disclosure herein. In certain example embodiments, sequencing includes next generation sequencing. In certain example embodiments, sequencing is multiplexed sequencing. In some embodiments, sequencing is performed on the sequencing library made of amplicons previously amplified in an amplification reaction.

[0077] Exemplary sequencing techniques include but are not limited to, single molecule Realtime (SMART) sequencing (see e.g., Eid et al., 2009, Science. 323(5910): 133-138), nanopore sequencing (see e.g., Bowden et al., 2019, Nature Comm. 10(1869), https: / / doi.org / 10.1038 / s41467-019-09637-5), massively parallel signature sequencing (MPSS) (see e.g., Brenner et al., 2000. Nature Biotech. 18(6):630-634) polony sequencing (see e.g., Shendure et al., 2009, Science. 309(5741): 1728-1732), 454 pyrosequencing (see e.g., Margulieset al. Nature. 437 (7057):376-380), Solexa / Illumina sequencing (see e.g., Bentley et al. Nature. 456(72318):53-59, Mardis et al., 2008. Annu rev Genom Hum Genet. 9:387-402), combinatorial probe anchor synthesis (cPAS) sequencing (see e.g., Drmanac et al. 2010, Science. 327 (5961): 78-81), SOLiD sequencing (see e.g., Valouev et al., 2008. Genome Res. 18(7): 1051-1063), Ion torrent semiconductor sequencing (see e.g., Rusk et al., 2011. Nature Methods. 8(1):44), DNA nanoball sequencing (see e.g., Porreca GJ. 2010. Nature Biotech. 28(l):43-44), heliscope single molecule sequencing (see e.g., Thompson et al., 2010. doi: 10.1002 / 0471142727. mb0710s92), microfluidic system-based sequencing (including but not limited to droplet based microfluidic sequencing methods and digital microfluidic sequencing methods (see e.g., Abate et al., 2013. Lab on a Chip 13(24) doi:10.1039 / c31c50905b, Pekin et al., 2011. Lab on a Chip 11(13):2156-2166; Fair et al., 2007. IEE Design & Test of Computers. 24(1): 10-24, Boles et al., 2011. Anal. Chem. 83(22):8439-8447, Zilionis et al., 2017, Nature Protocols. 12(l):44-73, Kan et al., 2004. Electrophoresis. 25 (21-22): 3564-3588, and others described elsewhere herein), tunneling currents sequencing (see e.g., Di Ventra. 2013. Nanotechnology. 24(34):342501, sequencing by hybridization (see e.g., Hanna et al., 2000. J. Clin. Microbiol. 38(7) :2715 -2721, Morey et al., 2013. Molecular Genetics and Metabolism. 110(l-2):3-24), sequencing with mass spectrometry (see e.g., Edwards et al., 2005. Mutation Research. 573(l-2):3-12, Hall et al. 2005. Analytical Biochem. 344(1): 53-69, Monforte et al. 1997. Nature Medicine 3(3):360-362, Beres et al. PNAS. 107(9):4371-4376), microscopy-based sequencing techniques (see e.g., Bell et al., 2012. Microscopy and Microanalysis) 18 (5): 1049-1053), and RNA polymerase-based sequencing (RNAP) (see e.g., Sasaki et al., Proc Natl Acad Sci U S A. 1998 Mar 31; 95(7): 3455-3460) and any combination thereof.

[0078] Sequencing data can be analyzed using various commercially and freely available tools prior to KDE analysis. Exemplary bioinformatic tools for sequencing analysis include but are not limited to bwa, samtools, PICARD, and freebayes. In some embodiments, custom scripts are employed for sequence data analysis. In some embodiments, sequencing data is analyzed according to the workflow shown in FIG. 9 and further discussed in the Working Examples. FIG. 9 shows a flow diagram of processing of the sequencing data. Vcf files can be generated from fastq files using open-source software BWA, SAMtools, PICARD, and freebayes. Custom scripts can then be used to analyze the vcf files. After sequence analysis, a Kemal Density Estimation (KDE)analysis can be performed to predict the fetal genotype as wild-type, homozygous for the autosomal recessive variant allele, or heterozygous for the autosomal variant allele. General principles of KDE analyses are generally known. KDE analysis generates result of the probability of a sample’s genotype (HOM / HET / WT).Target Genes and / or Variants

[0079] The method includes detecting target genes or target gene variants and / or detecting the probability that such a gene and / or variant is present in the maternal and / or fetal genome, which are also referred to herein as target genes of interest or variants of interest (VOI). It will be appreciated that the techniques and methods of the present disclosure can be applicable to any variant, particularly autosomal recessive gene (or allelic) variants. This disclosure and working examples herein provide exemplary non-limiting variants of interest. Additional target genes and / or variants of interest will be immediately appreciated by those of ordinary skill in the art and are within the spirit and scope of this disclosure. In some embodiments, the variant of interest is or contains one or more mutations, deletions, insertions, insertion / deletion (indels), or any combination thereof. In some embodiments, the mutation is a single nucleotide polymorphism. In some embodiments, the variant of interest directly or indirectly results in a disease, disorder, condition or a characteristic that is different than a wild-type or different variant. In some embodiments, the target gene and / or variant of interest is an autosomal recessive variant. In some embodiments, the method includes detecting the presence or absence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,I l l, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281,, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319,, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357,, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376,, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395,, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414,, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433,, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452,, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471,, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490,, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509,, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528,, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547,, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566,, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585,, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604,, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623,, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642,, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661,, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680,, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699,, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718,, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737,, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756,, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775,, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794,, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813,814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832,833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851,852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870,871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889,890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908,909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927,928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946,947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965,966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984,985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, to / or 1000 or more target genes and / or variants. In certain example embodiments, the one or more target genes and / or target variants are selected from the genes of Table 1.

[0080] In certain example embodiments, the method further includes sequencing one or more positive control spike-in fragmented DNA samples each corresponding to a 0, 1, or 2 copy number of one or more target genes.Samples

[0081] The samples can be biological samples. In some embodiments, the biological sample contains nucleic acids. In some embodiments, the biological sample is blood or a component thereof, such as plasma. In some embodiments, the nucleic acids include DNA, RNA or both. In some embodiments, the DNA in the sample is genomic DNA, circulating DNA (such as circulating fetal DNA (cfDNA), or both. In some embodiments, the genomic DNA is maternal genomic DNA. In some embodiments, the genomic DNA In certain example embodiments, the maternal genomic DNA, the cfDNA, or both are from blood or component thereof of the mother. In certain example embodiments, the blood or component thereof is plasma.

[0082] In some embodiments, the sample is processed or otherwise manipulated prior to sequences. In some embodiments nucleic acids are extracted from a sample. In some embodiments, DNA, RNA, or both are extracted. In some embodiments, genomic DNA is extracted. In some embodiments circulating DNA is extracted. In some embodiments, circulating fetal DNA is extracted from the sample. In some embodiments, both genomic DNA and circulating DNA is extracted from the sample. In some embodiments, both genomic DNA and circulating fetal DNA is extracted from the sample. In some embodiments, both maternal genomic DNA and circulating DNA is extracted from the sample. In some embodiments, both maternal genomic DNA and circulating fetal DNA is extracted from the sample. Exemplary methods of extracting genomic and circulating DNA, including circulating fetal DNA, from biological samples, including but not limited to blood or a component thereof (such as, but not limited to, plasma) are described in e.g., the Working Examples herein and are generally known to one of ordinary skill in the art. See e.g.,Chacon-Cortes, D., and Griffiths, L. Methods for extracting genomic DNA from whole blood samples: current perspectives. Journal of Biorepository Science for Applied Medicine . 2014;2: 1-9 https: / / doi.org / 10.2147 / BSAM.S46573; Cheli et al., Mol Biol Rep. 2020 Aug;47(8):6451-6455. doi: 10.1007 / sl 1033-020-05664-4; Andolfatto et al., European Respiratory Journal 2003 21 : 215- 219; DOI: 10.1183 / 09031936.03.00044303; Waters et al., J Vis Exp. 2013; (75): 4241; Lee et al., Precision cell-free DNA extraction for liquid biopsy by integrated microfluidics, npj Precision Oncology volume 4, Article number: 3 (2020); Van der Leest et al., Cancers (Basel). 2020 May; 12(5): 1222; Zhong et al., Prenat Diagn. 2022 Aug;42(9): l 173-1181; Pandoh et al., Biotechniques 66(2): 85-92, (2019); Perez-Barrios et al., Transitional Lung Cancer Res. 5(6): 665-672, 2016; and Pedini et al., Journal of Translational Medicine volume 19, Article number: 15 (2021). In some embodiments, nucleic acid extraction is performed using a commercially available machines, kits, and / or reagents, such as ThermoFisher Kingfisher FLEX machine, ThermoFisher MagMAX Cell- Free DNA Isolation kits and reagents, ThermoFisher GeneJET Whole Blood genomic DNA Purification Kit, ThermoFisher PureLink Genomic DNA Purification Kits, ThermoFisher MagMAX DNA Multi-Sample kit, and others, which will be known to one of ordinary skill in the art.

[0083] In some embodiments, the amount of nucleic acids extracted from the sample ranges from 1 to 1,1000 fg, pg, ng, pg, mg or more. In some embodiments, the amount of DNA, RNA, or both extracted from the sample ranges from 1 to 1,1000 fg, pg, ng, pg, mg or more. In some embodiments, the amount of genomic DNA extracted from the sample ranges from 1 to 1,1000 fg, pg, ng, pg, mg or more. In some embodiments, the amount of circulating DNA extracted from the sample ranges from 1 to 1,1000 fg, pg, ng, pg, mg or more. In some embodiments, the amount of circulating fetal DNA extracted from the sample ranges from 1 to 1,1000 fg, pg, ng, pg, mg or more. In some embodiments, the amount of genomic DNA and circulating fetal DNA extracted from the sample ranges from 1 to 1,1000 fg, pg, ng, pg, mg or more.

[0084] In some embodiments, the extracted nucleic acids are further processed prior to amplicon generation and / or sequencing. In some embodiments, the processing allows for determination of an epigenetic status of the DNA, such as methylation. For example, in some embodiments, the DNA is treated with bisulfite for bisulfite sequencing. See e.g., Li and Tollefsbol. Methods Mol Biol. 2011; 791: 11-21. In certain example embodiments, the methodfurther includes prior to sequencing and / or generating one or more amplicons, performing methylation sensitive digestion of the maternal gDNA, cfDNA, or both. In some embodiments, methylation sensitive digestion is performed using one or more methylation-sensitive restriction enzymes (MSREs). In general, MSREs cleave DNA at specific unmethylated-cytosine residues. Digested DNA can then be amplified and / or sequenced. As only the unmethylated DNA is digested by MSREs, the only amplification products detected are methylated DNA. Techniques and methods for methylation sensitive digestion of DNA will be immediately appreciated by one of ordinary skill in the art. See e.g., Zuo et al., Epigenomics. 2009 Dec 1; 1(2): 331-345; Fulnecek and Kovarik. BMC Genet. 2014; 15: 2; Harrison and Parle-McDermott. Front. Genet. 201 Vol. 2(11) https: / / doi.org / 10.3389 / fgene.2011.00074.METHODS OF TREATING AUTOSOMAL RECESSIVE DISORDERS

[0085] Described in certain example embodiments herein are methods of treating or preventing an autosomal recessive disorder or a symptom thereof in a fetus including detecting the presence of an autosomal recessive disorder in the fetus by performing a method of detecting the present disclosure described in greater detail elsewhere herein and treating or preventing the autosomal recessive disorder or a symptom thereof during gestation or post-partum.

[0086] In some embodiments, the treatment or prevention includes administering a therapeutic agent to treat or prevent the autosomal recessive disorder or symptom thereof in the fetus during gestation or in the neonate, child, or adult post-partum. Exemplary therapeutic agents include, but are not limited to polynucleotides, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, hormones, nutrients, minerals, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatories, anti-histamines, anti-infectives, chemotherapeutics, genetic modifying agents, and combinations thereof.

[0087] In some embodiments, the treating or preventing the autosomal recessive disorder or a symptom thereof comprises administering a genetic modifying agent to the fetus during gestation and / or to the neonate, child, or adult post-partum, where the genetic modifying agent is configured to modify the variant allele causative of the disease or disorder such that it is an allele variant that does not result in disease or disorder. Genetic modifying agents, include without limitation, CRISPR-Cas systems (Liu et al., Mol Cell. 2022 Jan 20;82(2):333-347; Anzalone et al., Nat Biotechnol. 2020 Jul;38(7):824-844. doi: 10.1038 / s41587-020-0561-9), transposases (Anzalone etal., Nat Biotechnol. 2020 Jul;38(7):824-844. doi: 10.1038 / s41587-020-0561-9); base editors (Anzalone et al., Nat Biotechnol. 2020 Jul;38(7):824-844. doi: 10.1038 / s41587-020-0561-9; Kantor et al., Int J Mol Sci. 2020 Aug 28;21(17):6240. doi: 10.3390 / ijms21176240; Newby and Liu. Mol Ther. 2021 Nov 3;29(11):3107-3124. doi: 10.1016 / j.ymthe.2021.09.002); Prime editing systems (see e.g., Chen and Liu. Nat Rev Genet. 2023 Mar;24(3):161-177. doi: 10.1038 / s41576- 022-00541-1; Newby and Liu. Mol Ther. 2021 Nov 3;29(11):3107-3124. doi: 10.1016 / j.ymthe.2021.09.002); Zinc Finger Nucleases (Shamshirgaran et al., Methods Mol Biol. 2022;2495:29-46. doi: 10.1007 / 978-l-0716-2301-5_2); TALENs (Shamshirgaran et al., Methods Mol Biol. 2022;2495:29-46. doi: 10.1007 / 978-1-0716-2301-5 2); RNAi (Setten et al., Nat Rev Drug Discov. 2019 Jun;18(6):421-446. doi: 10.1038 / s41573-019-0017-4; Traber and Yu. J Pharmacol Exp Ther. 2023 Jan;384(l): 133-154. doi: 10.1124 / jpet.122.001234). In some embodiments, one or more somatic cells is modified by the genetic modifying agent in the fetus or the neonate, child, or adult. In some embodiments, the genetic modifying agent does not modify a germline cell in the fetus, neonate, child, or adult.

[0088] In some embodiments, treating or preventing the autosomal recessive disorder or a symptom thereof comprises administering a gene therapy, RNA replacement, and / or protein replacement therapy (see e.g., Tan and Xu. Mol Cell Biochem. 2020 Nov;474(l-2):73-81. doi: 10.1007 / sl 1010-020-03834; High and Roncarolo. N Engl J Med. 2019 Aug l;381(5):455-464; Anguela and High. Annu Rev Med. 2019 Jan 27;70:273-288; Wolf et al., Nat Med. 2019 Jun;25(6):890-897. doi: 10.1038 / s41591 -019-0473-8; Peranteau and Flake. Mol Diagn Ther. 2020 Apr;24(2): 135-142. doi: 10.1007; Mendell et al., Mol Ther. 2021 Feb 3;29(2):464-488. doi: 10.1016; Sayed et al., Life Sci. 2022 Apr l;294: 120375. doi: 10.1016 / j .lfs.2022.120375; Vavilis et al. Pharmaceutics. 2023 Jan 3; 15(1): 166; and Leader et al., Nature Reviews Drug Discovery volume 7, pages 21-39 (2008) so as to mitigate and / or correct an RNA and / or protein deficiency caused by the detected variant allele to the fetus during gestation and / or to the neonate, child, or adult post-partum. In some embodiments, the gene therapy, RNA replacement, and / or protein replacement therapy is administered to a somatic cell.KITS

[0089] Described in several embodiments herein are kits that provide reagents for initial collecting and processing of samples and for use with the methods and assays of the presentdisclsoure. The kits can also provide reagents for storing samples for a period of time. The kit can include reagents for nucleic acid extraction, such as DNA extraction. The kit can include one or more reagents for In some embodiments, the kits also provide reagents, such as primers and other oligos, to incorporate barcodes or other unique molecular identifiers into the amplicon.

[0090] The kit may include one or more amplification reagents for performing amplification reactions. In some embodiments, the amplification reagents include, but are not limited to, dNTPs and / or nucleic acid primers used at any concentration appropriate for the amplification reaction, such as, but not limited to, a concentration of 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, or the like. The kit can include a polymerase for amplification. The polymerase can be any specific or general polymerase known in the art and useful, including Taq polymerase, Q5 polymerase, or the like.

[0091] The kit can include one or more primers and / or sets of primers for amplifying the nucleic acids. In some embodiments, the primers are configured to introduce one or more barcodes and / or unique molecular identifiers (UMIs) into the amplicon.

[0092] In some exemplary embodiments herein, the kits include amplification reagents and a set of primers. In certain example embodiments, a kit further includes a lysis reagent, such as a cell lysis reagent. In certain example embodiments, the kit includes a sample collection component. In certain example embodiments, the kit may comprise a reaction vessel including a pre-mixed combination of amplification reagents, such as primers, dNTPs, and / or polymerase. The pre-mixed combination can be ready to use or be formulated to be diluted, reconstituted, or otherwise prepared prior to use. In some embodiments, the pre-mixed combination is lyophilized. In such embodiments, the kit can include one or more reagents for reconstitution of the pre-mixed combination. In certain example embodiments the reaction vessel is designed to receive a sample or the sample collection component and be sealed. The initial amplification reaction on the individual sample can be done at the point-of-care in the reaction vessel containing the sample and sample collection component can be sent to a central processing facility for further processing, amplification, and / or sequencing according to the methods disclosed herein. In certain examples,embodiment the kit further includes a control sequence that can be amplified by the primers in the kit. The control may be included in the pre-mixed solution with the amplification reagents and the primer sets.Primers

[0093] In some embodiments, the kit includes one or more primers and / or primer sets. In some embodiments, the kit includes one or more primer sets where each primer set includes two or more primers. In some embodiments, two primers of the two or more primers of a primer set forms a primer pair. A primer pair refers to primers that are each capable of hybridizing to different sequences of a nucleic acid and are designed and configured to together define the region of the nucleic acid (e.g., DNA) that is amplified during an amplification reaction, such as a PCR or PCR- based amplification reaction. Any given primer set can have one, two, or more primer pairs. For example, a primer set having 6 primers, can include 1, 2, 3, or more primer pairs. It will be appreciated that a first primer can form a primer pair with more than one second primers. For example, a forward primer can form a primer pair with multiple different reverse primers. The primer pairs in primer sets having multiple primer pairs can be configured to amplify different and / or overlapping nucleic acid regions.

[0094] In some embodiments, the kit includes one or more primer sets each including two or more primers, which can be one or more primer pairs, where at least one primer of at least one of the one or more primer sets includes one or more barcodes, and where the one or more primer sets are configured to amplify one or more target sequences in one or more amplification steps from a sample in to generate amplicons that include the one or more target sequences and a unique combination of barcodes. In some embodiments, the barcoded primers present in the kit are included in the kit at equal amounts or concentrations. In some embodiments, each kit, each primer set, and / or each have a unique barcode or set of barcodes. In some embodiments, two or more barcodes in a kit, each primer set, and / or one or more primers are different from each other. In some embodiments, two or more barcodes in a kit, each primer set, and / or one or more primers are the same from other. In some embodiments, all primers and / or primer sets in a kit contain a barcode unique to that kit. In some embodiments, each primer set contains a barcode that is unique to each primer set. In some embodiments, each primer pair contains a barcode unique to that primer pair. In some embodiments, each primer contains a barcode unique to that primer.

[0095] In some embodiments, the kit includes at least one primer set that is configured for, PCR, loop-mediated isothermal amplification (LAMP) or reverse transcription loop-mediated isothermal amplification (RT-LAMP), SDA, RCA, LCR, RAM, NASBA, RPA, HAD, NEAR, NEAA, or MDA. In some embodiments, the primer set and / or primer pair includes a forward primer, a reverse primer, a forward inner primer (FIP), a backward inner primer (BIP), or any combination thereof. In some embodiments, one, two, or more primers in a primer set includes one or more barcodes. In some embodiments, the barcode(s) is / are inserted between the two targetspecific sequences of either the FIP, the BIP, or both.

[0096] In some embodiments, the kit contains one or more barcodes, such as in one or more primers of a primer set, that is / are unique to a first amplification and one or more barcodes, such as in one or more primers of a primer set that are unique to a second, third, fourth, fifth, etc. amplification reaction.

[0097] In some embodiments, the kit can include one or more primer sets that are configured for a PCR amplification, wherein one or more primers of the one or more primer sets configured for PCR amplification includes one or more PCR barcodes, sequencing adaptors, or both. In some embodiments, the one or more primer sets configured for PCR amplification contains a barcode unique to the PCR amplification primer sets and / or sequencing adaptors.

[0098] In some embodiments, the kit includes one or more primer sets configured for non- LAMP amplification by PCR. In some of these embodiments one or more primers of these one or more primer sets includes one or more PCR barcodes, sequencing adaptors, or both.

[0099] In some embodiments, a set of primers included in the kit includes two or more primer pairs and each primer pair includes a different barcode. In some embodiments, a single primer in the primer pair can have a barcode. In some embodiments, both primers in a primer pair can have a barcode, where the barcodes in each of the primers of the primer pair are the same or different. Each primer pair may amplify different or overlapping target sequences of a target polynucleotide. Example target polynucleotides are discussed in further detail below. Kits may be configured to detect a single target polynucleotide or more than one target polynucleotide so long as multiple primer sets are used for each target polynucleotide to incorporate a unique combination of barcodes into the resulting amplicons.

[0100] The number of barcoded primers to be included in a given kit is determined, in part, by the size of the barcode set for which individual barcodes can be pulled from and the number of samples that are to be pooled and run together. In certain example embodiment, the number of barcodes incorporated using the aforementioned primers pairs is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0101] Primer design is well known to those of skill in the art. In this regard, reference is made to Shi et al. Chin Sci Bull 48(12): 1165-1169 (2003); Chen et al. Analyst 145(2):440-444 (2020); Higgins et al. Bioinformatics 35(4):682-684 (2019); Liu et al. World J Microbiol Biotechnol 35(6):95 (2019); Jia et al. Front Microbiol 10:2860 (2019); Hardinge et al. Sci Rep 9(l):7400 (2019); Morabito et al. Mol Diagn Ther 17(3): 185-192 (2013); de Baar et al. J Clin Microbiol 37(6): 1813-1818 (1999); Lopez-Jimena et al. PLoS Negl Trop Dis 12(5):e0006381 (2018). The primers and barcodes may be further designed such that the do not form strong secondary structures or where the barcode has a GC content within a specified range.Barcodes

[0102] The term “barcode” as used herein refers to a short sequence of nucleotides (for example, DNA or RNA) that is used as an identifier for an associated molecule, such as a target molecule and / or target nucleic acid, or as an identifier of the source of an associated molecule, such as a cell-of-origin. A barcode may also refer to any unique, non-naturally occurring, nucleic acid sequence that may be used to identify the originating source of a nucleic acid fragment. The barcode sequence can provide a high-quality individual read of a barcode associated with a single subject, nucleic acid, target gene, and / or target variant, such that nucleic acids from multiple subjects, genes, and / or target variants can be sequenced together.

[0103] Barcoding may be performed based on any of the compositions or methods disclosed in patent publication WO 2014047561 Al, Compositions and methods for labeling of agents, incorporated herein in its entirety. In certain embodiments barcoding uses an error correcting scheme (T. K. Moon, Error Correction Coding: Mathematical Methods and Algorithms (Wiley, New York, ed. 1, 2005)). Not being bound by a theory, amplified sequences from single cells can be sequenced together and resolved based on the barcode associated with each cell.

[0104] A nucleic acid barcode can have a length of at least, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70,80, 90, or 100 nucleotides, and can be in single- or double-stranded form. Target molecule and / or target nucleic acids can be labeled with multiple nucleic acid barcodes in combinatorial fashion, such as a nucleic acid barcode concatemer. Typically, a nucleic acid barcode is used to identify a target molecule and / or target nucleic acid as being from a particular discrete volume, having a particular physical property (for example, affinity, length, sequence, etc.), or having been subject to certain treatment conditions. Target molecule and / or target nucleic acid can be associated with multiple nucleic acid barcodes to provide information about all of these features (and more).

[0105] As disclosed herein, unique nucleic acid identifiers are used to label the target molecules and / or target nucleic acids, for example origin-specific barcodes and the like. The nucleic acid identifiers, nucleic acid barcodes, can include a short sequence of nucleotides that can be used as an identifier for an associated molecule, location, or condition. In certain embodiments, the nucleic acid identifier further includes one or more unique molecular identifiers and / or barcode receiving adapters. A nucleic acid identifier can have a length of about, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 base pairs (bp) or nucleotides (nt). In certain embodiments, a nucleic acid identifier can be constructed in combinatorial fashion by combining randomly selected indices (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 indexes). Each such index is a short sequence of nucleotides (for example, DNA, RNA, or a combination thereof) having a distinct sequence. An index can have a length of about, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bp or nt. Nucleic acid identifiers can be generated, for example, by split-pool synthesis methods, such as those described, for example, in International Patent Publication Nos. WO 2014 / 047556 and WO 2014 / 143158, each of which is incorporated by reference herein in its entirety.

[0106] In certain example embodiments, barcodes are designed to avoid barcodes with sequence portions that are the reverse complement of a 3 prime end of a primer, such as the 3’ end of a FIP primer or a BIP primer used in isothermal amplifications embodiments disclosed herein.

[0107] One or more nucleic acid identifiers (for example a nucleic acid barcode) can be attached, or “tagged,” to a target molecule. This attachment can be direct (for example, covalent or noncovalent binding of the nucleic acid identifier to the target molecule) or indirect (for example, via an additional molecule). Such indirect attachments may, for example, include abarcode bound to a specific-binding agent that recognizes a target molecule. In certain embodiments, a barcode is attached to protein G and the target molecule is an antibody or antibody fragment. Attachment of a barcode to target molecules (for example, proteins and other biomolecules) can be performed using standard methods well known in the art. For example, barcodes can be linked via cysteine residues (for example, C-terminal cysteine residues). In other examples, barcodes can be chemically introduced into polypeptides (for example, antibodies) via a variety of functional groups on the polypeptide using appropriate group-specific reagents (see for example www.drmr.com / abcon). In certain embodiments, barcode tagging can occur via a barcode receiving adapter associated with (for example, attached to) a target molecule, as described herein.

[0108] Target molecules can be optionally labeled with multiple barcodes in combinatorial fashion (for example, using multiple barcodes bound to one or more specific binding agents that specifically recognizing the target molecule), thus greatly expanding the number of unique identifiers possible within a particular barcode pool. In certain embodiments, barcodes are added to a growing barcode concatemer attached to a target molecule, for example, one at a time. In other embodiments, multiple barcodes are assembled prior to attachment to a target molecule. Compositions and methods for concatemerization of multiple barcodes are described, for example, in International Patent Publication No. WO 2014 / 047561, which is incorporated herein by reference in its entirety.

[0109] In some embodiments, a nucleic acid identifier (for example, a nucleic acid barcode) may be attached to sequences that allow for amplification and sequencing (for example, SB S3 and P5 elements for Illumina sequencing). In certain embodiments, a nucleic acid barcode can further include a hybridization site for a primer (for example, a single-stranded DNA primer) attached to the end of the barcode. For example, an origin-specific barcode may be a nucleic acid including a barcode and a hybridization site for a specific primer. In particular embodiments, a set of originspecific barcodes includes a unique primer specific barcode made, for example, using a randomized oligo type NNNNNNNNNNNN.

[0110] Labeled target molecules and / or target nucleic acids associated origin-specific nucleic acid barcodes (optionally in combination with other nucleic acid barcodes as described herein) can be amplified by methods known in the art, such as polymerase chain reaction (PCR). For example,the nucleic acid barcode can contain universal primer recognition sequences that can be bound by a PCR primer for PCR amplification and subsequent high-throughput sequencing. In certain embodiments, the nucleic acid barcode includes or is linked to sequencing adapters (for example, universal primer recognition sequences) such that the barcode and sequencing adapter elements are both coupled to the target molecule. In particular examples, the sequence of the origin specific barcode is amplified, for example using PCR. In some embodiments, an origin-specific barcode further includes a sequencing adaptor. In some embodiments, an origin-specific barcode further includes universal priming sites. A nucleic acid barcode (or a concatemer thereof), a target nucleic acid molecule (for example, a DNA or RNA molecule), a nucleic acid encoding a target peptide or polypeptide, and / or a nucleic acid encoding a specific binding agent may be optionally sequenced by any method known in the art, for example, methods of high-throughput sequencing, also known as next generation sequencing or deep sequencing. A nucleic acid target molecule labeled with a barcode (for example, an origin-specific barcode) can be sequenced with the barcode to produce a single read and / or contig containing the sequence, or portions thereof, of both the target molecule and the barcode. Exemplary next generation sequencing technologies include, for example, Illumina sequencing, Ion Torrent sequencing, 454 sequencing, SOLiD sequencing, and nanopore sequencing amongst others. In some embodiments, the sequence of labeled target molecules is determined by non-sequencing based methods. For example, variable length probes or primers can be used to distinguish barcodes (for example, origin-specific barcodes) labeling distinct target molecules by, for example, the length of the barcodes, the length of target nucleic acids, or the length of nucleic acids encoding target polypeptides. In other instances, barcodes can include sequences identifying, for example, the type of molecule for a particular target molecule (for example, polypeptide, nucleic acid, small molecule, or lipid). For example, in a pool of labeled target molecules containing multiple types of target molecules, polypeptide target molecules can receive one identifying sequence, while target nucleic acid molecules can receive a different identifying sequence. Such identifying sequences can be used to selectively amplify barcodes labeling particular types of target molecules, for example, by using PCR primers specific to identifying sequences specific to particular types of target molecules. For example, barcodes labeling polypeptide target molecules can be selectively amplified from a pool, thereby retrieving only the barcodes from the polypeptide subset of the target molecule pool.[OHl] A nucleic acid barcode can be sequenced, for example, after cleavage, to determine the presence, quantity, or other feature of the target molecule. In certain embodiments, a nucleic acid barcode can be further attached to a further nucleic acid barcode. For example, a nucleic acid barcode can be cleaved from a specific-binding agent after the specific-binding agent binds to a target molecule or a tag (for example, an encoded polypeptide identifier element cleaved from a target molecule), and then the nucleic acid barcode can be ligated to an origin-specific barcode. The resultant nucleic acid barcode concatemer can be pooled with other such concatemers and sequenced. The sequencing reads can be used to identify which target molecules were originally present in which discrete volumes.

[0112] In some embodiments, the origin-specific barcodes are reversibly coupled to a solid or semisolid substrate. In some embodiments, the origin-specific barcodes further comprise a nucleic acid capture sequence that specifically binds to the target nucleic acids and / or a specific binding agent that specifically binds to the target molecules. In specific embodiments, the origin-specific barcodes include two or more populations of origin-specific barcodes, wherein a first population includes the nucleic acid capture sequence and a second population includes the specific binding agent that specifically binds to the target molecules. In some examples, the first population of origin-specific barcodes further includes a target nucleic acid barcode, wherein the target nucleic acid barcode identifies the population as one that labels nucleic acids. In some examples, the second population of origin-specific barcodes further includes a target molecule barcode, wherein the target molecule barcode identifies the population as one that labels target molecules.

[0113] A nucleic acid barcode may be cleavable from a specific binding agent, for example, after the specific binding agent has bound to a target molecule. In some embodiments, the originspecific barcode further includes one or more cleavage sites. In some examples, at least one cleavage site is oriented such that cleavage at that site releases the origin-specific barcode from a substrate, such as a bead, for example a hydrogel bead, to which it is coupled. In some examples, at least one cleavage site is oriented such that the cleavage at the site releases the origin-specific barcode from the target molecule specific binding agent. In some examples, a cleavage site is an enzymatic cleavage site, such an endonuclease site present in a specific nucleic acid sequence. In other embodiments, a cleavage site is a peptide cleavage site, such that a particular enzyme cancleave the amino acid sequence. In still other embodiments, a cleavage site is a site of chemical cleavage.

[0114] In some embodiments, the target molecule is attached to an origin-specific barcode receiving adapter, such as a nucleic acid. In some examples, the origin-specific barcode receiving adapter includes an overhang and the origin-specific barcode includes a sequence capable of hybridizing to the overhang. A barcode receiving adapter is a molecule configured to accept or receive a nucleic acid barcode, such as an origin-specific nucleic acid barcode. For example, a barcode receiving adapter can include a single-stranded nucleic acid sequence (for example, an overhang) capable of hybridizing to a given barcode (for example, an origin-specific barcode), for example, via a sequence complementary to a portion or the entirety of the nucleic acid barcode. In certain embodiments, this portion of the barcode is a standard sequence held constant between individual barcodes. The hybridization couples the barcode receiving adapter to the barcode. In some embodiments, the barcode receiving adapter may be associated with (for example, attached to) a target molecule. As such, the barcode receiving adapter may serve as the means through which an origin-specific barcode is attached to a target molecule. A barcode receiving adapter can be attached to a target molecule according to methods known in the art. For example, a barcode receiving adapter can be attached to a polypeptide target molecule at a cysteine residue (for example, a C-terminal cysteine residue). A barcode receiving adapter can be used to identify a particular condition related to one or more target molecules, such as a cell of origin or a discreet volume of origin. For example, a target molecule can be a cell surface protein expressed by a cell, which receives a cell-specific barcode receiving adapter. The barcode receiving adapter can be conjugated to one or more barcodes as the cell is exposed to one or more conditions, such that the original cell of origin for the target molecule, as well as each condition to which the cell was exposed, can be subsequently determined by identifying the sequence of the barcode receiving adapter / barcode concatemer.

[0115] In some embodiments, an origin-specific barcode further includes a capture moiety, covalently or non-covalently linked. Thus, in some embodiments the origin-specific barcode, and anything bound or attached thereto, that include a capture moiety are captured with a specific binding agent that specifically binds the capture moiety. In some embodiments, the capture moiety is adsorbed or otherwise captured on a surface. In specific embodiments, a targeting probe islabeled with biotin, for instance by incorporation of biotin- 16-UTP during in vitro transcription, allowing later capture by streptavidin. Other means for labeling, capturing, and detecting an originspecific barcode include: incorporation of aminoallyl-labeled nucleotides, incorporation of sulfhydryl-labeled nucleotides, incorporation of allyl- or azide-containing nucleotides, and many other methods described in Bioconjugate Techniques (2ndEd), Greg T. Hermanson, Elsevier (2008), which is specifically incorporated herein by reference. In some embodiments, the targeting probes are covalently coupled to a solid support or other capture device prior to contacting the sample, using methods such as incorporation of aminoallyl-labeled nucleotides followed by 1- Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) coupling to a carboxy-activated solid support, or other methods described in Bioconjugate Techniques. In some embodiments, the specific binding agent has been immobilized for example on a solid support, thereby isolating the origin-specific barcode.

[0116] Software for DNA barcoding can use integration of a field information management system (FIMS), laboratory information management system (LIMS), sequence analysis tools, workflow tracking to connect field data and laboratory data, database submission tools and pipeline automation for scaling up to eco-system scale projects. Geneious Pro can be used for the sequence analysis components, and the two plugins made freely available through the Moorea Biocode Project, the Biocode LIMS and Genbank Submission plugins handle integration with the FIMS, the LIMS, SynBio software system, workflow tracking and database submission. Additionally, other barcoding designs and tools have been described (see e g., Birrell et al., (2001) Proc. Natl Acad. Sci. USA 98, 12608-12613; Giaever, et al., (2002) Nature 418, 387-391; Winzeler et al., (1999) Science 285, 901-906; and Xu et al., (2009) Proc Natl Acad Sci U S A. Feb 17; 106(7):2289-94), all of which can be adapted for use with the present disclosure particularly for designing and interpreting barcodes and barcoding data.Amplification Reagents

[0117] In some embodiments, the kit includes one or more amplification reagents. Amplification reagents and systems known in the art can be designed for use with the methods and systems detailed herein. In some embodiments, amplification is isothermal. Any suitable RNA or DNA amplification technique may be used. In certain example embodiments, the RNA or DNA amplification is an isothermal amplification. In certain example embodiments, the isothermalamplification may be nucleic-acid sequence-based amplification (NASBA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HD A), or nicking enzyme amplification reaction (NEAR). In certain example embodiments, non-isothermal amplification methods may be used which include, but are not limited to, PCR, multiple displacement amplification (MDA), rolling circle amplification (RCA), ligase chain reaction (LCR), or ramification amplification method (RAM). In particular embodiments a first amplification method is utilized, for example an isothermal amplification, and a second amplification method is utilized subsequently, for example, a non -isotherm al amplification method. In certain embodiments when two amplification methods are used, the amplification methods are the same, in other embodiments, the amplification methods are different. In an aspect, at least one of the amplification methods is an isothermal amplification that retains one or more barcodes in the amplicon. In an aspect, at least one of the amplification methods comprise amplifying a plurality of pooled samples. The isothermal amplification kits may be designed, via the selection of appropriate polymerases and buffers, to work over a wide range of temperatures.Lysis Reagents

[0118] In some embodiments, the method includes cell lysis, such as to obtain cellular nucleic acids. In some embodiments, the kit contains one or more cell lysis reagents in order to break open or lyse a cell for analysis of the materials therein. Cell lysis reagents include, but are not limited to, a detergent, a salt as described above, such as NaCl, KC1, ammonium sulfate [(NH^SCL], or others. Detergents that may be appropriate for the compositions and methods of the present disclosure can include Triton X-100, sodium dodecyl sulfate (SDS), CHAPS (3-[(3- cholamidopropyl)dimethylammonio]-l-propanesulfonate), ethyl trimethyl ammonium bromide, nonyl phenoxypoly ethoxylethanol (NP-40), Polysorbate 10, Polysorbate 20, Proteinase K, Saponin. Concentrations of detergents may depend on the particular application, and may be specific to the reaction in some cases. Additional cell lysis and nucleic acid extraction reagents, such as those suitable for obtaining genomic DNA and cfDNA present in blood are described elsewhere herein and will be appreciated by those of ordinary skill in the art in view of the disclosure herein.Additional Reagents

[0119] The kits can also include additional reagents, such as protein inhibitors, reaction indicators capable of providing a detectable signal of reaction status and / or result, suitable control reagents, combinations thereof, and the like. In some embodiments, the reaction indicator is a colorimetric or turbidimetric indicator. In some embodiments, the reaction indicator is a pH sensitive indicator. In some embodiments, the control reagents are capable of amplifying a control target in a sample. Suitable control targets include housekeeping targets, or other targets that are expected to be expressed or are otherwise capable of being detected in all samples tested. In some embodiments, the suitable control targets are also typically expressed or otherwise capable of detection at about the same level or amount or at about the same relative level or relative amount (such as relative to sample weight or volume on a total, liquid, total protein, total nucleic acid, total DNA, total RNA, or dry matter basis).Sample Collection Component

[0120] The kit can include and / or the method can employ, in some embodiments, a sample collection component. The sample collection component may comprise one or more components to be used by a patient or health care worker. The sample collection component may include a syringe, needle, catheter, and / or the like configured to draw or obtain blood of blood component form the subject. The sample collection may include a nasopharyngeal swab, oropharyngeal swab, or other swab for collection of an oral or nasal sample. Sterile polyester or nylon swabs with aluminum or plastic shafts are preferable. Squirt bottles, bulbs, syringes or other means for rinsing of an oral or nasal cavity can be provided as a sample collection component. The bulb, bottle, or syringe may be provided pre-loaded with saline for the collection of a specimen. A sterile suction catheter suction apparatus can be provided for obtention of a nasopharyngeal / nasal aspirate. Jars and other sample containers can be used for collection of stool samples, and may further include additional probes, swabs and tubes for further sample processing. In some embodiments, the sample collection component is configured for collection of a nasal swab, an oral swab, a nasal wash, an oral wash, a fecal sample, a wound swab, or a combination thereof.

[0121] Sample collection components may comprise a vial, tube or other containment means for the collected sample. In some embodiments the sample collection component can contain a preservative, an anti-coagulant, or other enzyme or other composition suitable for collection and / orpreservation of a blood sample or a blood component sample. The sample collection component may comprise tubes, vials, containers or other receptacles for the collection of a sample wash, for example, nasal or oral washes. The containment means may comprise a lid configured to receive all or a portion of a swab, or may be configured such that the swab is provided as a portion of the lid to the containment means. The containment may comprise one or more reagents, including amplification reagents, solvents, detergents and other solutions, and can be designed for use in further sample manipulations, shipping, and subsequent reactions. In a preferred embodiment, one or more of the sample collection components is resistant to heat, and the collected sample can be further reacted and processed within the sample collection component, for example, for conducting the isothermal amplification reaction. Sample collection means may be further provided with ice packs and other shipping packaging.

[0122] In some embodiments, the sample collection component can be or include a sample dosing component. The sample dosing component can be configured to portion out or separate out a portion a collected sample to a desired amount (e.g., a dose appropriate for the downstream reaction(s)) and optionally facilitate placement of the portion in a reaction or other collection vessel. Non-limiting sample dosing components are spoons, spatulas, depression sticks, droppers, capillaries, syringes, and the like. In some embodiments, the sample dosing component can be part of or form the sample collection component. In some embodiments, the sample dosing component and the sample dosing component are separate components of the kit.Reaction Vessel

[0123] The reaction vessel may be one of the sample collection components described above, or may be a separate vessel suitable for the storing and shipping of a sample. The reaction vessel may comprise pre-mixed ingredients, including amplification reagents, detergents, sterile solutions, and other reagents that may be utilized in further sample processing. In certain embodiments, the reaction vessel is configured to receive a sample collection component above, such as a swab. As an example, the reaction vessel may comprise a lid configured to receive all or a portion of a swab, or may be configured such that the swab is provided as a portion of the lid of the reaction vessel. The reaction vessel may be pre-loaded with sterile solution for the storing of the sample. More than one reaction vessel may be included if more than one target is to be detected. Each reaction vessel can comprise reagents specific for the detection of each separate target.Reagents may be provided lyophilized, with reconstitution at the point of use, for example with a solution provided as a separate element of the kit. The reaction vessel is preferably designed to be heated and can be configured for use with particular heating elements and / or type of end-user.

[0124] The reaction vessel may be suitable for processing of individual samples or may comprise multi-well plates capable of processing multiple samples simultaneously.

[0125] In some embodiments, the reaction vessel includes a pre-mixed combination of amplification reagents and barcoded primers and configured to be sealed after receiving the sample, sample collection component, sample dosing component, or a combination thereof.

[0126] In some embodiments, the reaction vessel is configured for use in an isothermal amplification reaction. In some embodiments, the reaction vessel is configured for use at a point of care. In some embodiments, the reaction vessel is configured for use in an isothermal amplification reaction conducted at a point of care.

[0127] Further embodiments are illustrated in the following Examples which are given for illustrative purposes only and are not intended to limit the scope of the invention.EXAMPLES

[0128] Now having described the embodiments of the present disclosure, in general, the following Examples describe some additional embodiments of the present disclosure. While embodiments of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit embodiments of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.Example 1

[0129] Amniocentesis and chorionic villi sampling (CVS) are still considered the gold standard for diagnostic prenatal testing, but they still carry a high risk of miscarriage, are expensive, and must be conducted in a tertiary care setting. Accordingly, the demand for noninvasive sampling techniques has increased substantially in recent years. One such option that is already widely clinically implemented is known as cell-free noninvasive prenatal testing or screening (NIPT / NIPS).

[0130] The presence of cell-free fetal DNA (cffDNA) in the maternal circulation was discovered in 1997, through the extraction and amplification of Y chromosomal (fetal) DNA from samples from pregnant women (Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, Wainscoat JS. Presence of fetal DNA in maternal plasma and serum. Lancet. 1997;350(9076):485-7. Epub 1997 / 08 / 16. doi: 10.1016 / 80140-6736(97)02174-0. PubMed PMID: 9274585). The subsequent introduction of massive parallel sequencing enabled using cffDNA analysis as an NIPT - by combining shotgun sequencing with the counting of cffDNA fragments, the relative over- or underrepresentation of chromosomes can be estimated (Chiu RW, et al.,. Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing ofDNA in maternal plasma. Proc Natl Acad Sci U S A. 2008;105(51):20458-63. Epub 20081210 and Guseh SH. Noninvasive prenatal testing: from aneuploidy to single genes. Hum Genet. 2020; 139(9): 1141-8. Epub 20190925). NIPT is currently mainly used for the detection of common aneuploidies such as trisomy 13, 18 and 21, and has a higher sensitivity and specificity compared to the traditional maternal serum screening (Guseh SH. Noninvasive prenatal testing: from aneuploidy to single genes. Hum Genet. 2020;139(9):l 141-8. Epub 20190925). Cell-free NIPT rapidly evolved to become one of the standard components of prenatal care, and it is estimated that already more than 6 million women have undergone NIPT pregnancy screening (Guseh SH. Noninvasive prenatal testing: from aneuploidy to single genes. Hum Genet. 2020;139(9):l 141-8. Epub 20190925 and Bianchi DW, Chiu RWK. Sequencing of Circulating Cell-free DNA during Pregnancy. N Engl J Med. 2018;379(5):464-73).

[0131] As mentioned above, the principal application at present for NIPT is the detection of potential fetal chromosomal abnormalities (i.e., the common aneuploidies and to some extent smaller copy number variants (CNVs)). However, aside from aneuploidies and CNVs, many birthdefects are caused by a monogenic disorder. Hence, the scope of NIPT has expanded in recent years to also include some single-gene diseases. However, because cffDNA is mostly placental in origin, complicating factors such as confined placental mosaicism are not as much an issue for single-gene testing compared to noninvasive aneuploidy and CNV screening.

[0132] Population-wide carrier screening has been proven a very cost-effective measure to reduce severe autosomal recessive conditions since its implementation in the 1970’s (Nelson WB, Swint JM, Caskey CT. An economic evaluation of a genetic screening program for Tay-Sachs disease. Am J Hum Genet. 1978;30(2): 160-6 and Rowley PT, Loader S, Kaplan RM. Prenatal screening for cystic fibrosis carriers: an economic evaluation. Am J Hum Genet. 1998;63(4): 1160- 74). It also has been reported that about 60% of all severe postnatal single-gene disorders follow an autosomal dominant inheritance pattern, mostly caused by de novo mutations (Yang Y, et al. Molecular findings among patients referred for clinical whole-exome sequencing. JAMA. 2014;312(18): 1870-9. doi: 10.1001 / jama.2014.14601). A novel noninvasive screening test based on cffDNA sequencing was recently developed and marketed by Baylor Genetics, and includes a panel of 30 causative genes for common dominant monogenic disorders (Zhang J, et al., Noninvasive prenatal sequencing for multiple Mendelian monogenic disorders using circulating cell- freefetal DNA. NatMed. 2019;25(3):439-47. Epub 20190128). This novel NIPT approach (named PreSeek) was designed specifically for paternally inherited or de novo mutations in genes associated with conditions such as Noonan syndrome, Cornelia de Lange syndrome, and Osteogenesis imperfecta, which taken together reach a fairly high cumulative prevalence (Zhang J, et al., Non-invasive prenatal sequencing for multiple Mendelian monogenic disorders using circulating cell-free fetal DNA. Nat Med. 2019;25(3):439-47. Epub 20190128). PreSeek’s methodology is based on a unique molecular barcoding for sequencing analysis combined with a SNP -based fetal fraction determination, allowing for an accurate and high-quality test result (Zhang J, et al., Non-invasive prenatal sequencing for multiple Mendelian monogenic disorders using circulating cell-free fetal DNA. Nat Med. 2019;25(3):439-47. Epub 20190128).

[0133] There are two key analytical approaches to detecting monogenic diseases including the relative mutation dosage (RMD) and relative haplotype dosage (RHDO). The RMD requires parental genotyping and design of mutation assay. The RHDO requires information of the parental haplotype and, more specifically, the determination of the informative heterozygous SNPs linkedto the mutation site. In RMD the comparison is made between counts at the specific mutation site; in RHDO, haplotypes are compared in the maternal plasma using multiple SNPs, thereby increasing the accuracy and reproducibility.

[0134] Carrier screening with reflex single-gene NIPS has also been developed to screen for a small number of autosomal recessive conditions such as cystic fibrosis, hemoglobinopathies, and spinal muscular atrophy via next generation sequencing (NGS). It is possible to identify a paternally inherited fetal variant that is absent in the maternal genome, as seen in cystic fibrosis transmembrane conductance regulator (CFTR) protein screening, or identify variants in the fetus that are not present in the maternal genome by investigating polymorphic regions using parental haplotypes as a reference.14 The implementation of this testing is beneficial in cases where pregnancy is unplanned, late gestational prenatal carrier testing, the partner is unavailable for testing, and / or to expand the accessibility of testing for qualified, uninsured patients (Jenkins LA, Deans ZC, Lewis C, Allen S. Delivering an accredited non-invasive prenatal diagnosis service for monogenic disorders and recommendations for best practice. PrenatDiagn. 2018;38(l):44-51. doi: 10.1002 / pd.5197).

[0135] Because the bulk of all cell-free DNA (cfDNA) in the mother’s circulation is of maternal origin, dominant conditions caused by a paternally inherited or de novo mutation are the most straightforward to test cffDNA for, as is the case for the PreSeek test. Screening for autosomal recessive and X-linked disorders is more challenging, as it requires assessing whether the maternal allele has been passed on to the fetal genome or not.15 The inherited allele is genetically identical to the maternal allele, lowering its fetal detection. It is nevertheless feasible, and multiple reports have already been published (Jenkins LA, Deans ZC, Lewis C, Allen S. Delivering an accredited non-invasive prenatal diagnosis service for monogenic disorders and recommendations for best practice. PrenatDiagn. 2018;38(l):44-51. doi: 10.1002 / pd.5197; Cutts A, Vavoulis DV, Petrou M, Smith F, Clark B, Henderson S, Schuh A. A method for noninvasive prenatal diagnosis of monogenic autosomal recessive disorders. Blood. 2019; 134(14): 1190-3. Epub 20190823; and Hayward J, Chitty LS. Beyond screening for chromosomal abnormalities: Advances in non- invasive diagnosis of single gene disorders and fetal exome sequencing. Semin Fetal Neonatal Med. 2018;23(2):94-101. Epub 20180102.)

[0136] While NIPT, including those utilizing cffDNA, for paternally inherited monogenic conditions is already in clinical practice, it is not yet applicable to the maternally inherited conditions. This Example describes and demonstrates NIPT assays for maternally inherited allelic variants.

[0137] The NIPT for maternally inherited allelic variants described and demonstrated in this Example. In general, the NIPT for maternally inherited allelic variants uses amplicon-next generation sequencing (NGS) to measure the allele imbalance of a target mutation. The maternal genotype or variant allele frequency (VAF) is determined by amplicon-NGS using maternal genomic DNA. The genotype VAF of the cfDNA is determined by the same amplicon-NGS using cell-free DNA (cfDNA). The allele imbalance is determined from these two values. The sequencing data are bioinformatically analyzed and fetal genotype is predicted by analyzing fetal fraction and allele imbalance. This NIPT for maternally inherited allelic variants needs only maternal peripheral blood, meaning only a blood draw from the mother is needed to collect an appropriate sample. Cell-fee DNA is extracted from the plasma. Hemolysis can be prevented during the sample collection and / or preparation by utilizing Streck tubes. Primer pools utilized for amplicon-NGS can include primer sets for detection of control and target SNPs. PCR performed for amplicon-NGS can be multiplex and suitable barcodes and adaptors can be incorporated into the primers and / or amplicons for NGS and post-sequencing data deconvolution. This test can therefore estimate the Mendelian inheritance pattern of a variant-of-interest (VOI), particularly of a maternally inherited VOI, in fetuses during pregnancies.Targeted mutation analysis for couples at risk for rare autosomal recessive conditions.

[0138] In the Middle Eastern population, it was determined it might be necessary to utilize an alternative approach to assist couples at-risk for rare autosomal recessive conditions. This approach would be helpful for a couple who have had a previous child diagnosed with a rare recessive condition who wish prenatal diagnosis. The non-invasive approach can be performed earlier in the pregnancy (>9 weeks). Quantitative PCR was used to determine the zygosity of the mutation targeted mutation. Three specific primers were used in two qPCR reactions: fm+rv and fw+rv, including a forward primer targeting the mutant allele (fm), a forward primer targeting the wildtype allele (fw), and a reverse primer (rv). Alternatively, quantification was done by droplet digital PCR, a quantitative method of performing digital PCR based on water / oil emulsion droplettechnology. This method was used to amplify specific variants by amplicon based NGS and was compared with the results with capture NGS. The comparison demonstrated 100% concordance.

[0139] Next, as a proof-of-concept, three distinct populations of data distribution regarding variant allele fraction (VAF) in maternal plasma where the VOI (e.g., SMN1) in the maternal genomic DNA (gDNA) is heterozygous and an amplicon-NGS based assay to determine the fetal VOI (e.g., SMN 1) copy numbers was developed and tested.

[0140] When both mother and father carry the same heterozygous variant, theoretically, the variant allele fraction (VAF) in maternal plasma is affected by whether the VOI allele is passed to the fetus. However, the degree of the change is expected to be small, since fetal cfDNA in maternal plasma is the minority. Hence, paternal allele frequency (PAF) plays a key role in accurate estimation of the inheritance phase of the fetus. By expression of a ratio of the change in VAF (AVAF) compared to PAF, the cut-off values to determine homozygosity of wild-type and variant can be established. Amplicon NGS was applied to improve NGS accuracy of a VOI. Fetal Fraction (FF) was calculated by a selection of quality control (QC) SNPs from PreSeek trio / solo NIPTs. In addition to the maternal VAF (VAF), the paternal allele fraction (PAF) may also be obtained using this NIPT for maternally inherited allele variants of this Example.

[0141] Nine reported cases with FF between 10% and 20% were selected Table 2. Three primer sets for 3 QC SNPs in different chromosomes (4, 6, and 18) were designed (Table 3). A QC SNP from one of the 3 QC SNPs in each case generated the PAF value. PCR was triplicated and performed on plasma cfDNA and maternal gDNA, respectively. In this proof-of-concept, paternal gDNA was not required since the genotyping had been obtained from previous conventional NGS. Prior to NGS, three amplicons in each sample were pooled. The sequencing data were processed by NextGENe. The results were presented as plots as function of VAF or AVAF / PAF.Results

[0142] The pattern of 3 populations can be observed by single-run data points. The relationship among plasma VAF, maternal VAF, and fetal zygosity can be described as: Plasma VAF = maternal VAF*(l-ff) + fetal VAF*n*ff n = fetal mutant alleles:• n=0: WT (assuming wild-type)• n=l : HET (assuming heterozygous variant)• n=2: HOM (assuming homozygous variant)

[0143] Three populations of plasma VAF (n = 0, 1, 2) was expected based on Mendelian inheritance. The preliminary run (without duplicate or triplicate) showed three populations (fetal WT, fetal HET, and fetal HOM) (FIG. 1), but a population sitting between fetal HOM and fetal HET was also observed. It is suspected this was due to the experimental variation, and it was further investigated by doing experiments in triplicate from the PCR amplification step.

[0144] Triplicate PCR with 3 sets of primers was performed for 9 pairs of plasma cfDNA / maternal gDNA in two independent experiments. 14 informative data points in which maternal gDNA were heterozygous were collected. For each data point, AVAF, which is plasma VAF subtracted by maternal VAF, was normalized to PAF, obtained from conventional NGS (PreSeek solo / trio tests) (FIG. 2A-2B). As expected, 3 populations assumptively corresponding to fetal HOM, fetal HET, and fetal WT are distinct as shown in FIG. 2B. The mean values of these 3 groups are 1.038, -0.1613, and -1.265, respectively, which are close to the theoretical values (1,0, and -1). Besides, it was attempted to get PAF values from amplicon NGS (Table 4).

[0145] This Example at least demonstrates establishment of a methodology to visualize three clusters that represent fetal zygosity in the background of maternal heterozygous variants which is interpreted as a successful proof-of-concept which can be applied to other VOI.Example 2

[0146] The Pre Seek test currently only detects SNVs and / or small indels. SMN1 CNV is a common mutation in carrier screening. This Example describes and demonstrates design and preliminarily testing of an amplicon-NGS based assay to determine the fetal SMN1 copy numbers.

[0147] The SMN1 copy number of the plasma DNA (plasma_cn) can be described in this equation: plasma_cn=mat_cn*(l-ff) +fetal_cn*ff

[0148] Assuming the maternal DNA copy number (mat_cn, usually 2 or 1) is known, if the fetal fraction (ff) and plasma DNA copy number (plasma_cn) can be calculated, the SMN1 copy number of fetal DNA can be determined.

[0149] A multiplex PCR amplicon-NGS based assay was designed to calculate the ff and plasma copy number. PCR was used to amplify the SMN1 gene (as a proof of concept example gene), prepare NGS sequencing libraries using the PCR product, run sequencing, and count the NGS reads. If the PCR reaction did not reach a plateau, the read counts were correlated to the gene copy number. The amplicon includes the c.840 position, so the reads from SMN1 and SMN2 can be differentiated. This can demonstrate that choice of amplicon can be used to differentiate between genes with sequence similarity. An additional 13 pairs of primers were also designed to amplify common SNPs. These 13 amplicons have two purposes: (1) VAFs of the variants are used to calculate the fetal fraction; (2) the NGS reads counts are used as the internal controls for SMN1 NGS reads normalization then the plasma DNA copy number can be calculated.MethodsPrimers

[0150] To efficiently amplify short cfDNAs, the amplicons were designed to be aboutlOObp (98-101bp). All primers were first tested using gDNA and plasma DNA individually and then combined to a final concentration of 5 pM for multiplex PCR. 10 ng DNA (plasma DNA or fragmented gDNA) and 15 cycles were used for multiplex PCR. After cleanup, NGS adaptors were added to the PCR products to prepare the sequencing libraries. Libraries were co-sequenced with other NGS samples with target data size of 50-100 Mb per sample. NGS data were analyzed using NextGen software and home-made scripts.Samples

[0151] Plasma DNA was used to test the assay. Then spike-in fragmented DNA samples were used as the positive samples. DNAs are fragmented to 150-200bp before PCR. We have two series spike-in samples: 1) spike-in a 0 copy SMN1 sample into a normal 2 copy SMN1 sample with different rations; 2) spike-in a 1 copy SMN1 sample into a normal 2 copy SMN1 samples. So, we have 10 mixture samples with different spike-in content (ff) and different mixture SMN1 copy numbers (between 0 to 2, and between 1 to 2). Table 4 lists the mixture samples info, including mix ratio, expected spike-in level (fetal fraction) and expected mixture SMN1 copy number. Samples 1 and samples 6 are 2 copies sample only and will be used as normalization reference control.Results

[0152] From the VAFs of variants from 13 amplicons, we can calculate the fetal fraction (ff). We compared the calculated ff with the expected ff (spike-in level), and the values are close and highly corelated. The results are shown in FIG. 3. Further details of the two groups are listed in Table 4Mixture SMN1 copy number (plasma copy number)

[0153] The NGS reads of all amplicons were counted. For SMN1, reads belonging to SMN1 using the unique c.840 were the only ones counted. First, the SMN1 reads counts were normalized to other amplicons in the same sample. This was followed by normalizing the values with a sample with known SMN1 copy number in the batch / run. Mixture 1 and 6 both have 2 copies of SMN1. The calculated SMN1 copy number was then compared with the expected values. These values were close and corelated, as shown in FIG. 4.Fetal DNA SMN1 copy number

[0154] Using the equation described above, we calculated the fetal DNA SMN 1 copy numbers. The expected SMN1 copy number for Series 1 samples are 0 and for Series 2 are 1. The results are showed in FIG. 5. The calculated copy numbers of series 2 samples are smaller than expected (1 copy) but are consistent in a defined range.Discussion

[0155] This Example at least demonstrates design and testing of a multiplex PCR / amplicon- NGS based assay to calculate the ff, the plasma SMN1 copy number, and the SMN1 copy number of fetal DNA. Using spike-in fragmented DNA, it was demonstrated that SMN1 copy number of fetal DNA can be calculated from amplicon-NGS. The robustness of this assay can be further optimized by: 1) add more amplicons to improve the fetal fraction calculation; 2) adjust multiplex PCR conditions to minimize the effect of difference in PCR efficiency.Example 3

[0156] An Autosomal Recessive (AR)-NIPT assay using amplicon-based NGS method and a fetal fraction-based data analysis algorithm was developed and demonstrated in e.g., Examples 1 and 2 herein. Applicant processed and analyzed 20 samples with invasive results ( N=3 batches). The preliminary results identified limitations of the fetal fraction-based analysis and indicated that the data analysis algorithm could further be optimized. This Example at least describes and demonstrates a kernel density estimation (KDE) method for data analysis to predict fetal genotype. Kernel density estimation (KDE) is a statistical tool to study the data distribution and to group data. KDE analysis generates result of the probability of a sample’s genotype (HOM / HETAVT). Methods

[0157] Only maternal peripheral blood was needed and was collected. Cell-free DNA (cfDNA)ZgDNA was extracted from plasma / WBC in a single tube of blood using methods described in Stray et al., Stray J, Zimmermann B. Isolation of Cell-Free DNA from Maternal Plasma. Methods Mol Biol. 2019;1885:309-323. doi: 10.1007 / 978-l-4939-8889-l_21. PMID: 30506207. Briefly, plasma was separated from the peripheral blood of pregnant women at about 10-12 weeks of pregnancy. Cell-free DNA (cfDNA) was extracted from plasma using ThermoFisher Kingfisher FLEX machine following the manufacturer’s instructions. Next multiplexed PCR amplification followed by NGS sequencing of PCR amplicons was performed with a panel of primers that included common SNPs (as a control) and a target mutation. For each sample, a pair of primers was designed for the specific mutation. Primers for a group of 91 common SNPs was also designed, which were used as internal controls. About 3-5 ng cfDNA was used for multiplex PCR. FastStart Taq polymerase from Roche was used for PCR amplification accordingto the manufacturer’s suggested conditions. About 10 ng of PCR product was ligated with a seqencing adaptor with an index sequence using Roche KAPA HyperPrep Kits following manufacture’s protocol. After multiplex PCR amplification and addition ofNGS adaptors, libraries were subjected to sequencing. The NGS library was sequenced on an Illumina Novaseq 6000 with 2X100bp read length. Custom primers were designed for each individual mutation based on patient family information. Sequencing data were processed using appropriate open-source bioinformatic tools (bwa, samtools, PICARD, freebayes) and custom scripts. FIG. 9 shows a flow diagram of processing of the sequencing data. Vcf files were generated from fastq files using open-source software BWA, SAMtools, PICARD, and freebayes. Custom scripts are used to analyze the vcf files. Statistical analysis using Kernel density estimation (KDE) model was used to generate the probability of the fetal DNA genotype.

[0158] For the AR-NIPT assay, multiplex PCR of 91 common SNPs and 1 target mutation were run for each sample. Depending on the genotypes of MAT cfDNA and fetal cfDNA, any plasma_cfDNA variant is in one of 7 groups (Table 5). For AR-NIPT, groups 3-5 (MAT cfDNA is HET) were focused on.

[0159] After KDE analysis of all plasma variants, 91 SNPs and 1 mutation are grouped, and the genotype of fetal DNA can be determined (FIG. 6). KDE results for all samples is shown in Table 6

[0160] The mutation PCR should have the similar performance as common SNPs in the multiplex PCR s a proposition in this model. In the scenario that a mutation PCR is not compatible to the common SNPs PCR performance, this variant could not be used by this algorithm. “Mutation not compatible” will be noted for this kind of cases. For example, VAF of the sample (Table 7) indicates that the amplification efficiency of mutant allele is far from WT allele frequency, thisindicates a different performance comparing to common SNP PCR, and KDE analysis cannot be performed.Categorizing the KDE results

[0161] All 3 groups are normal distribution and there may be overlapping between 2 distributions, so a variant has the probability of 2 genotypes from data analysis.• For a variant if the probability of minor content is >10%, the result is considered low confidence and named with probability of 2 groups (FIG. 7 at “A”).• Result with >90% probability of major content is considered High confidence and is name with major content (FIG. 7 at “B”).KDE analysis results summary (HOM / HET / WT):KDE analysis generates result of the probability of a sample’s genotype (HOM / HET / WT). For 20 samples, 4 have mutations that are not compatible with KDE analysis, 10 samples have the genotype with high confidence, and 6 with low confidence (Table 8).Clinical analysis results summary (HOM vs Non HOM)

[0162] For clinical purpose, both WT and HET are negative samples and immediate clinical attention may not be needed. After we group the samples to positive (HOM) and negative (non HOM), we have 3 samples with low confidence results (Table 7).Summary

[0163] Compared to the fetal fraction-based algorithm of Examples 1 and 2, the KDE-based data analysis in this algorithm of this Example uses more information generated from the NGS and provides a more quantitative method to analyze the data.Example 4

[0164] This Example details validation of the assays demonstrated in Examples 1-3 across a more heterogenous population. Examples 1-3 established a multiplex PCR / amplicon-NGS based assay to calculate the ff, the plasma SMN1 copy number, and the SMN1 copy number of fetal DNA. It was also demonstrated that genotype of fetal DNA can be calculated from amplicon-NGS using spike-in fragmented DNA. Furthermore, the data analysis algorithm was optimized using a KDE-based method. These Examples were performed with samples validated within a fairly homogeneous population from the Middle East. This Example details the expansion of the validation pool to a generalized U.S. population.

[0165] For this 500 participants will be recruited from U.S. hospitals and thus representative of a heterogenous population. Participants will be 18 years of age or older, pregnant with singleton babies only. Participants will be excluded if they are expecting twins, triplets, any multiple baby pregnancy, and if they are diagnosed with any active cancer. Then the fetal genotype will be predicted using methods described in relation to Examples 1-3 and as detailed in the present Example.Methods cfDNA Extraction

[0166] Plasma will be separated from the peripheral blood of 500 pregnant women at 10-12 weeks of pregnancy. Cell-free DNA (cfDNA) is extracted from plasma using Thermofisher Kingfisher FLEX machine following the manufacturer’s instructions.PCR and Next Generation Sequencing

[0167] For each sample, a pair of primers will be designed for the VOI mutation. Primers will also be designed for a group of 92 common SNPs, which will be used in all samples as controls. 3-5ng cfDNA is used for multiplex PCR. Multiplex PCR will be run using all 93 pairs of primers. The PCR product will be ligated with an adaptor with an index sequence using conventional techniques. After PCR amplification, the library will be sequenced on an Illumina Novaseq 6000. Data Processing and Analysis

[0168] Vcf files will be generated from fastq files using open-source software BWA andFreebayes. Custom scripts will also be used to analyze the vcf files and call the fetal genotypes.Example 5

[0169] With additional data collected on the general U.S. population from Example 4, a statistical model for normalization of the baseline probability readings (WT), followed with machine learning models to automatically generate optimized cut-off thresholds to separate HET and HOM calls for the KDE analysis will be generated.Methods

[0170] Samples from recruitment in Aim 1 (generalized U.S. population) will be divided into a 70% training population and a 30% testing population. The baseline normalization and machine learning models will be trained and optimized on former data set, with the models tested on the latter. The receiver operating characteristic (ROC) curves and their area under curve (AUC) scores will be calculated and compared between the training and testing population to evaluate the generalization ability of our methodology.Optimization of genotype calling

[0171] The detection of affected pregnancies will also be optimized by employing multiple modes of reference genomes in the statistic algorithm. By utilizing a diverse set of reference genomes, we can account for population-specific and locus high-resolution genomic copy number variations (CNV) data and reduce the likelihood of false-positive or false-negative results in our cfDNA-based prenatal screening tests. Ultimately, the integration of multiple reference genomes will not only improve the performance of the prenatal screening tests but also contribute to the development of more inclusive and reliable diagnostic tools, allowing for better-informed clinical decision-making, timely interventions, and improved patient outcomes across different populations.

[0172] To enhance the detection of affected pregnancies, technical replicates will be incorporated into the testing workflows. By minimizing random fluctuations and biases in quantitative multiplex PCR and next-generation sequencing (NGS), an increase accuracy and reproducibility is expected. This includes performing triplicate multiplex PCR of cfDNA and NGS sequencing steps. This approach will boost statistical power by reducing variability introduced during library preparation and sequencing, leading to more accurate detection. Additionally, thismethodological improvement will decrease the need for repeating assays due to failed libraries, thereby improving practical utility and saving time.Example 6

[0173] A novel noninvasive screening test, PreSee assayk, based on cffDNA sequencing was recently developed and marketed by Baylor Genetics, and includes a panel of 30 causative genes for common dominant monogenic disorders (Zhou AE, Travassos MA. Bringing Sickle-Cell Treatments to Children in Sub-Saharan Africa. New England Journal of Medicine. 2022;387(6):488-91). This NIPT approach was designed specifically for paternally inherited or de novo mutations in genes associated with conditions such as Noonan syndrome, Cornelia de Lange syndrome and Osteogenesis imperfecta, which taken together reach a fairly high cumulative prevalence (Zhou AE, Travassos MA. Bringing Sickle-Cell Treatments to Children in Sub-Saharan Africa. New England Journal of Medicine. 2022;387(6):488-91). PreSeek assay’s methodology is based on a unique molecular barcoding for sequencing analysis combined with a SNP-based fetal fraction determination, allowing for an accurate and high-quality test result (Zhou AE, Travassos MA. Bringing Sickle-Cell Treatments to Children in Sub-Saharan Africa. New England Journal of Medicine. 2022;387(6):488-91). This Example details addressing some of the most common ARDs, as listed in Table 9 with the assay of the present disclosure.

[0174] The study participants will be: 1) couples with an increased risk of having a pregnancy affected by an ARD because both parents were found to be carriers or 2) couples with a previously affected child opting for screening for a new pregnancy.Sample Collection

[0175] Maternal plasma samples from at least 500 families with known variants will be obtained and tested. These samples will be collected from patients Emory Health and Baylor College of Medicine and / or other locations as appropriate. Participants will be 18 years of age or older; they will be excluded if they have any active cancer or expecting twins, triplets, etc. Maternal blood samples will be collected into Cell-free DNA Streck tubes, transported to the lab and cffDNA will be extracted as soon as the samples are received at the laboratory.Fetal fraction determination

[0176] Fetal fraction values will be obtained using a method previously published by White and colleagues, which is based on the specific methylation detection of RASSF1A (Hoskovec J,Hardisty EE, Talati AN, Carozza JA, Wynn J, Riku S, ten Bosch JR, Vora NL. Maternal carrier screening with single-gene NIPS provides accurate fetal risk assessments for recessive conditions. Genetics in Medicine. 2023;25(2)). Since the RASSF1A gene is hypermethylated in fetal cfDNA but not in maternal cfDNA, qPCR detection of RASSFlA’s differential methylation status using both cfDNA digested with methylation-sensitive restriction enzymes (ctl) and cfDNA without restriction enzymes digestion (ct2) will give the fetal fraction value (FIG. 8).NGS and data analysis methodology

[0177] Amplicon NGS methodology will be used to detect and confirm known parental variants. This method uses oligonucleotide probes designed to target and amplify specific regions of interest within a gene, followed by NGS.

[0178] NGS Full Gene Target Capture will be used for screening of new pregnancies for which the carrier status of the parents is unknown but had a previously affected pregnancy or child. With targeted sequencing, a subset of genes or genomic regions are specifically isolated and sequenced. The targeted capturing and enrichment step utilizes hybridization to target-specific biotinylated probes, which are subsequently isolated by magnetic pull-down. A clinically validated method to achieve this (Zhou AE, Travassos MA. Bringing Sickle-Cell Treatments to Children in Sub- Saharan Africa. New England Journal of Medicine. 2022;387(6):488-91).

[0179] After sequencing (either amplicon or full gene capture), analysis will be done by a custom data analysis pipeline which includes in-house built data processing scripts and commonly used software packages such as BWA-GATK-Mutect2 or Illumina DRAGEN Bio-IT platform.Mutation analysis

[0180] A published statistical method will be used to determine the mutation and the zygosity status of the mutations (Zhou AE, Travassos MA. Bringing Sickle-Cell Treatments to Children in Sub-Saharan Africa. New England Journal of Medicine. 2022;387(6):488-91). A group of samples with known fetal fraction, fetal mutation and fetal mutation type (WT / HET / HOM) will be used to train a statistical model proposed in this publication. The sample mutation and zygosity status will be calculated using NGS data and fetal fraction.Confirmation of positive finding:

[0181] Quantitative PCR will be used to confirm the mutation detected by NGS and to determine the zygosity of the mutation. Three specific primers will be used in and fw+rv, includinga forward primer targeting the mutant allele (fm), a forward primer targeting the wildtype allele (fw), and a reverse primer (rv). Alternatively, quantification will be done by droplet digital PCR, a quantitative method of performing digital PCR based on water / oil emulsion droplet technology. Confirmation of NGS findings will be performed prior to clinical reporting.

[0182] Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth.

[0183] Further attributes, features, and embodiments of the present invention can be understood by reference to the following numbered aspects of the disclosed invention. Reference to disclosure in any of the preceding aspects is applicable to any preceding numbered aspect and to any combination of any number of preceding aspects, as recognized by appropriate antecedent disclosure in any combination of preceding aspects that can be made. The following numbered aspects are provided:1. A method of detecting autosomal recessive variant alleles and / or autosomal recessive disorders in a fetus, the method comprising: sequencing one or more regions of maternal genomic DNA (gDNA) comprising one or more target genes or one or more target gene variants, or both, of a mother of the fetus to determine a maternal genotype variant allele frequency (VAF); sequencing one or more regions of circulating fetal DNA (cfDNA) comprising the one or more target genes in a biological sample obtained from the mother to determine a fetal genotype VAF; anddetecting an allele imbalance from a maternal genotype VAF and the fetal genotype VAF thereby determining a genotype and a presence or absence of an autosomal recessive variant allele and / or disorder in the fetus.2. The method of aspect 1, wherein detecting an allele imbalance comprises determining a copy number of the maternal genotype VAF and the fetal genotype VAF.3. The method of any one of aspects 1-2, wherein determining an allele imbalance comprises performing a Kernel density estimation (KDE) analysis that comprises comparing a probability of the genotype of the maternal genotype VAF, fetal genotype VAF, or both to groups corresponding to a homozygous genotype, a heterozygous genotype, and a wild type genotype prepared by Kernel Density Estimation (KDE) analysis of a reference database for each of the one or more target genes.4. The method of any one of aspects 1-3, wherein sequencing comprises next generation sequencing.5. The method of any one of aspects 1-4, wherein sequencing is multiplexed sequencing.6. The method of any one of aspects 1-5, wherein sequencing comprises generating one or more amplicons via polymerase chain reaction and sequencing the one or more amplicons.7. The method of any one of aspects 1 -6, further comprising performing methylation sensitive digestion of the maternal gDNA, cfDNA, or both prior to sequencing, generating one or more amplicons, or both.8. The method of any one of aspects 1-7, wherein the one or more target genes are selected the group consisting of SMN1, ACADVL, ADA2, ADAT3, AGL, AK2, ALDH7A1, ARSA, ASL, B3GALT6, C12orf57, Ca2, CANT1, CEP290, CTFR, CNPY3, C0G6, COL6A2, CUP2U1, DLD, EPCAM, FARS2, FKRP, GAA, GALC, GALNS, GBA, GLB1, GNPAT, GNPTG, GRM7, GUSB, HBB, HEXB, HIBCH, HSPG2, INVS, ISCA2, KIAA0586, LAMA1, LAMA2, LARGE1, LEPRE1, LIFR, MAN1B1, METTL5, MMUT, MUT, NDUFS4, NKX6-2, NPC1, NPHP3, NRAP, OTC, PCCA, PGAP3, PI4KA, PKHD1, PLA2G6, POLR1C, RAB3GAP1, RAG1, RAG2, RNASEH2C, SCO2, SUOX, SURF1, TBCE, TTN, WDR62, WNT7A.9. The method of any one of aspects 1-8, wherein the one or more target gene variants are selected from the group consisting of: ACADVL c.65C>A,p.Ser22Ter; ACADVL c, 134C>A, p.Ser45*; ACADVL c,1048G>A, p.Gly35OArg / c,134C>A, p.Ser45*; ACADVL c, 134C>A,р.Ser45*; ADA2 c.882-2A>G; ADAT3 c.430G>A,p.Vall44Met; AGL c.294-2A>T; AK2 с.524G>C,p.Argl75Pro; ALDH7A1 c.518-2A>C; ARSA c.H08-2A>G; ASL c,1060C<T, p.Gln354*; B3GALT6 c.77T>C, p.Leu26Pro; C12orf57 c. lA>G,p.MetlVal; Ca2 c.232+lG>A; CANT1 c. 894_898dupGCCGC, p.Ser303Alafs*21; CANT1 c.902_906dupGCGCC, р.Ser303fs*21; CFTRc.l418del; CFTRp.Gly473Glufs*54; CNPY3 c.496-3_496-2delCA; C0G6 с, 1167-24A>G; COL6A2 c.2422+lG>A; COL6A2 C.1817-1G>A; CYP2U1 c.947A>T,p.Asp316Val; DLD c.685G>T,p.Gly229Cys; EPCAM c.583dup; EPCAM c.499dup, р.Glnl67Profs*21; EPCAM c.367G>C,p.Glyl23Arg; EPCAM c.451G>C; FARS2 с.431A>G,p.Tyrl44Cys; FKRP C.941OT, p.Thr314Met; FKRP C.1364OA, p.Ala455Asp; GAA c. l657C>T,p.Q553*; GAA c,1657C>T,p.Q553X; GALC c,1886G>T,p.W629L; GALNS c.958G>A, p.Glu320Lys; GALNS c.970G>C, p.Ala324Pro; GBA c. 1448T>C,p.Leu483Pro; GLB1 c.622C>T,p.Arg208Cys; GNPAT c.569-3T>G; GRM7 c.461T>C ,p.Ilel54Thr; GUSB c. l832G>A,p.Arg611Gln; HBB c.315+lG>A; HEXB c.94C>T, p.Gln32*; HIBCH c.410C>T, p.Alal37Val; HSPG2 c.l 1674+1G>T; INVS c,1807C>T, p.Arg603*; ISCA2 c.229G>A, р.Gly77Ser; KIAA0586 c.4191del , p.Argl397fs; LAMA1 c.6779C>A, p.Ser2260Tyr; LAMA2 с.3924+2T>C; LAMA2 c,1762del,p.Ala588Leufs*l l; LARGE1 exon 5-7 deletion; LEPRE1 c.1839-2A>C; LIFR c.1258C>T, p.Gln420Ter; MAN1B 1 c.1376del,p.Gly459Alafs* 12; METTL5 c.541+lG>C; MMUT c.810_811delins,p.Ala271Leufs*l l; MUT c.785G>T,p.Ser262Ile; NDUFS4 c.350+lG>A; NKX6-2 c,196delC,p.R66fs*122; NPC1 c.2130+lG>A; NPC1 c.3609_3610delAC, p.L1204YfsX53; NPHP3 c.3675dupT,p.Alal226fs; NPHP3 c.3674_3675insA; NRAP c.400_407del, p.Cysl34Serfs*12; OTC c.275G>A; p.Arg92Gln; PCCA c.l l l_130del,p.Tyr391Asnfs*5; PCCA c. 425G>A,p.Glyl42Asp; PGAP3 c.850C>T; р.His284Tyr; PI4KA c.3O53C>T ,p.SerlO18Leu; PKHD1 c.9374C>G, p.Ser3125*; PKHD1 с.2180A>G,p.Asn727Ser; PLA2G6 c.2070_2072del; p.Val691del; PLA2G6 c,1933C>T; р.Arg645*; POLR1C c.934T>C, p.Ser312Pro; RAB3GAP1 c,1009C>T,p.Arg337*; RAG1 с.555del,p.Lysl86Serfs*15; RAG2 c, 1332C>G, p.Ile444Met; RNASEH2C c.202C>G,p.Leu68Val; SCO2 c.2T>C / p.Metl; SMN1 deletion of exons 7 and 8; SUOX c.520del; p.Aspl74Thrfs*13; SURF1 c.758_759del,p.Thr253Serfs*38; TBCE c,155_166dell2bp, p.S52_G55del; TTN c.35054del,p.Hisl l685Leufs*15; WDR62 c.758_760delACA, p.Asn253del; and WNTA c.874C>T,p.Arg292Cys.10. The method of any one of aspects 1-9, further comprising sequencing one or more positive control spike-in fragmented DNA samples each corresponding to a 0, 1, or 2 copy number of the one or more target genes.11. The method of any one of aspects 1-10, wherein the maternal genomic DNA, the cfDNA, or both are from blood or component thereof of the mother.12. The method of aspect 11, wherein the blood or component thereof is plasma.13. A method of treating or preventing an autosomal recessive disorder or a symptom thereof in a fetus comprising: detecting a presence of an autosomal recessive disorder in the fetus by performing a method of any one of aspects 1-12; and treating or preventing the autosomal recessive disorder or a symptom thereof during gestation or post-partum.14. The method of aspect 13, wherein treating or preventing comprises administering a genetic modifying agent to the fetus during gestation and / or to a neonate, child, or adult post-partum, where the genetic modifying agent is configured to modify the variant allele causative of the autosomal recessive disease or disorder such that it is an allele variant that does not result in autosomal recessive disease or disorder.15. A kit compri sing : one or more primer sets, wherein each primer set is configured to amplify a region of a target gene of an autosomal recessive variant allele and / or autosomal recessive disorder and are further configured for use in a method of any one of aspects 1-12.16. The kit of aspect 15, wherein the target gene is selected from the group consisting of: SMN1, ACADVL, ADA2, ADAT3, AGL, AK2, ALDH7A1, ARSA, ASL, B3GALT6, C12orf57, Ca2, CANT1, CEP290, CTFR, CNPY3, COG6, COL6A2, CUP2U1, DLD, EPCAM, FARS2, FKRP, GAA, GALC, GALNS, GBA, GLB1, GNPAT, GNPTG, GRM7, GUSB, HBB, HEXB, HIBCH, HSPG2, INVS, ISCA2, KIAA0586, LAMA1, LAMA2, LARGE1, LEPRE1, LIFR, MAN IB 1, METTL5, MMUT, MUT, NDUFS4, NKX6-2, NPC1, NPHP3, NRAP, OTC, PCCA, PGAP3, PI4KA, PKHD1, PLA2G6, POLR1C, RAB3GAP1, RAG1, RAG2, RNASEH2C, SCO2, SUOX, SURF1, TBCE, TTN, WDR62, WNT7A, or any combination thereof.17. The kit of any one of aspects 15-16, wherein the target gene is a target gene variant selected from the group consisting of: ACADVL c.65OA,p.Ser22Ter; ACADVL C.134 A, p.Ser45*; ACADVL c, 1048G>A, p.Gly35OArg / c.1340 A, p.Ser45*; ACADVL C.134 A, p.Ser45*; ADA2 c.882-2A>G; ADAT3 c.430G>A,p.Vall44Met; AGL c.294-2A>T; AK2 c.524G>C,p.Argl75Pro; ALDH7A1 c.518-2A>C; ARSA c.H08-2A>G; ASL c,1060C<T, p.Gln354*; B3GALT6 c.77T>C, p.Leu26Pro; C12orf57 c.lA>G,p.MetlVal; Ca2 c.232+lG>A; CANT1 c. 894_898dupGCCGC, p.Ser303Alafs*21; CANT1 c.902_906dupGCGCC, р.Ser303fs*21; CFTRc.l418del; CFTRp.Gly473Glufs*54; CNPY3 c.496-3_496-2delCA; C0G6 с.1167-24 A>G; COL6A2 c.2422+lG>A; COL6A2 C.1817-1G>A; CYP2U1 c.947A>T,p.Asp316Val; DLD c.685G>T,p.Gly229Cys; EPCAM c.583dup; EPCAM c.499dup, р.Glnl67Profs*21; EPCAM c.367G>C,p.Glyl23Arg; EPCAM c.451G>C; FARS2 с.431A>G,p.Tyrl44Cys; FKRP C.941OT, p.Thr314Met; FKRP C.1364 A, p.Ala455Asp; GAA c. l657C>T,p.Q553*; GAA c,1657C>T,p.Q553X; GALC c,1886G>T,p.W629L; GALNS c.958G>A, p.Glu320Lys; GALNS c.970G>C, p.Ala324Pro; GBA c. 1448T>C,p.Leu483Pro; GLB1 c.622C>T,p.Arg208Cys; GNPAT c.569-3T>G; GRM7 c.461T>C ,p.Ilel54Thr; GUSB c. l832G>A,p.Arg611Gln; HBB c.315+lG>A; HEXB C.94 T, p.Gln32*; HIBCH C.410 T, p.Alal37Val; HSPG2 c.l 1674+1G>T; INVS C.1807 T, p.Arg603*; ISCA2 c.229G>A, р.Gly77Ser; KIAA0586 c.4191del , p.Argl397fs; LAMA1 C.6779 A, p.Ser2260Tyr; LAMA2 с.3924+2T>C; LAMA2 c,1762del,p.Ala588Leufs*l 1; LARGE1 exon 5-7 deletion; LEPRE1 c.1839-2A>C; LIFR c.1258OT, p.Gln420Ter; MAN1B 1 c.1376del,p.Gly459Alafs* 12; METTL5 c.541+lG>C; MMUT c.810_811delins,p.Ala271Leufs*l l; MUT c.785G>T,p.Ser262Ile; NDUFS4 c.350+lG>A; NKX6-2 c,196delC,p.R66fs*122; NPC1 c.2130+lG>A; NPC1 c.3609_3610delAC, p.L1204YfsX53; NPHP3 c.3675dupT,p.Alal226fs; NPHP3 c.3674_3675insA; NRAP c.400_407del, p.Cysl34Serfs*12; OTC c.275G>A; p.Arg92Gln; PCCA c.l l l_130del,p.Tyr391Asnfs*5; PCCA c. 425G>A,p.Glyl42Asp; PGAP3 C.850 T; р.His284Tyr; PI4KA C.3053 T ,p.SerlO18Leu; PKHD1 c.9374C>G, p.Ser3125*; PKHD1 с.2180A>G,p.Asn727Ser; PLA2G6 c.2070_2072del; p.Val691del; PLA2G6 C.1933OT; р.Arg645*; P0LR1C c.934T>C, p.Ser312Pro; RAB3GAP1 c,1009C>T,p.Arg337*; RAG1 с.555del,p.Lysl86Serfs*15; RAG2 c,1332C>G, p.Ile444Met; RNASEH2C c.202C>G,p.Leu68Val; SC02 c.2T>C / p.Metl; SMN1 deletion of exons 7 and 8; SUOX c.520del;p.Aspl74Thrfs*13; SURF1 c.758_759del,p.Thr253Serfs*38; TBCE c,155_166dell2bp, p.S52_G55del; TTN c.35054del,p.Hisl l685Leufs*15; WDR62 c.758_760delACA, p.Asn253del; and WNTA c.874OT,p.Arg292Cys, or any combination thereof.

[0184] 18. The kit of aspect 15, further comprising one or more reagents for carrying out polymerase chain reaction, sequencing, or both.

Claims

CLAIMSWhat is claimed is:

1. A method of detecting autosomal recessive variant alleles and / or autosomal recessive disorders in a fetus, the method comprising: sequencing one or more regions of maternal genomic DNA (gDNA) comprising one or more target genes or one or more target gene variants, or both, of a mother of the fetus to determine a maternal genotype variant allele frequency (VAF); sequencing one or more regions of circulating fetal DNA (cfDNA) comprising the one or more target genes in a biological sample obtained from the mother to determine a fetal genotype VAF; and detecting an allele imbalance from a maternal genotype VAF and the fetal genotype VAF thereby determining a genotype and a presence or absence of an autosomal recessive variant allele and / or disorder in the fetus.

2. The method of claim 1, wherein detecting an allele imbalance comprises determining a copy number of the maternal genotype VAF and the fetal genotype VAF.

3. The method of claim 2, wherein determining an allele imbalance comprises performing a Kernel density estimation (KDE) analysis that comprises comparing a probability of the genotype of the maternal genotype VAF, fetal genotype VAF, or both to groups corresponding to a homozygous genotype, a heterozygous genotype, and a wild type genotype prepared by Kernel Density Estimation (KDE) analysis of a reference database for each of the one or more target genes.

4. The method of claim 1, wherein determining an allele imbalance comprises performing a Kernel density estimation (KDE) analysis that comprises comparing a probability of the genotype of the maternal genotype VAF, fetal genotype VAF, or both to groups corresponding to a homozygous genotype, a heterozygous genotype, and a wild type genotype prepared by KDE analysis of a reference database for each of the one or more target genes or one or more target gene variants.

5. The method of claim 1, wherein sequencing comprises next generation sequencing.

6. The method of claim 1, wherein sequencing is multiplexed sequencing.

7. The method of claim 1, wherein sequencing comprises generating one or more amplicons via polymerase chain reaction and sequencing the one or more amplicons.

8. The method of claim 7, further comprising performing methylation sensitive digestion of the maternal gDNA, cfDNA, or both prior to sequencing, generating one or more amplicons, or both.

9. The method of claim 1, further comprising performing methylation sensitive digestion of the maternal gDNA, cfDNA, or both prior to sequencing.

10. The method of claim 1, wherein the one or more target genes are selected the group consisting of: SMN1, ACADVL, ADA2, ADAT3, AGL, AK2, ALDH7A1, ARSA, ASL, B3GALT6, C12orf57, Ca2, CANT1, CEP290, CTFR, CNPY3, COG6, COL6A2, CUP2U1, DLD, EPC AM, FARS2, FKRP, GAA, GALC, GALNS, GBA, GLB 1, GNPAT, GNPTG, GRM7, GUSB, HBB, HEXB, HIBCH, HSPG2, INVS, ISCA2, KIAA0586, LAMA1, LAMA2, LARGE1, LEPRE1, LIFR, MAN1B1, METTL5, MMUT, MUT, NDUFS4, NKX6-2, NPC1, NPHP3, NRAP, OTC, PCCA, PGAP3, PI4KA, PKHD1, PLA2G6, POLR1C, RAB3GAP1, RAG1, RAG2, RNASEH2C, SCO2, SUOX, SURF1, TBCE, TTN, WDR62, WNT7A.

11. The method of claim 1, wherein the one or more target gene variants are selected from the group consisting of: ACADVL c.65OA,p.Ser22Ter; ACADVL C.134OA, p.Ser45*; ACADVL c, 1048G>A, p.Gly350Arg / C.134OA, p.Ser45*; ACADVL c, 134C>A, p.Ser45*; ADA2 c.882- 2A>G; ADAT3 c.430G>A,p.Vall44Met; AGL c.294-2A>T; AK2 c.524G>C,p.Argl75Pro; ALDH7A1 c.518-2A>C; ARSA c.H08-2A>G; ASL c,1060C<T, p.Gln354*; B3GALT6 c.77T>C, p.Leu26Pro; C12orf57 c.lA>G,p.MetlVal; Ca2 c.232+lG>A; CANT1 c. 894_898dupGCCGC, p.Ser303Alafs*21; CANT1 c.902_906dupGCGCC, p.Ser303fs*21; CFTR c, 1418del; CFTR p.Gly473Glufs*54; CNPY3 c.496-3_496-2delCA; COG6 c.l 167-24A>G; COL6A2 c.2422+lG>A; COL6A2 C.1817-1 G>A; CYP2U1 c.947A>T,p.Asp316Val; DLD c.685G>T,p.Gly229Cys; EPCAM c.583dup; EPCAM c.499dup, p.Glnl67Profs*21; EPCAM c.367G>C,p.Glyl23Arg; EPCAM c.451G>C; FARS2 c.431A>G,p.Tyrl44Cys; FKRP c.941C>T, р.Thr314Met; FKRP c,1364C>A, p.Ala455Asp; GAA c, 1657OT,p.Q553*; GAA с. l657C>T,p.Q553X; GALC c,1886G>T,p.W629L; GALNS c.958G>A, p.Glu320Lys; GALNS c.970G>C, p.Ala324Pro; GBA c. 1448T>C,p.Leu483Pro; GLB1 c.622C>T,p.Arg208Cys; GNPAT c.569-3T>G; GRM7 c.461T>C ,p.Ilel54Thr; GUSB c, 1832G>A,p.Arg611Gln; HBB c.315+lG>A; HEXB c.94C>T, p.Gln32*; HIBCH c.410C>T, p.Alal37Val; HSPG2 C.1 1674+1G>T; INVS c,1807C>T, p.Arg603*; ISCA2 c.229G>A, p.Gly77Ser; KIAA0586 c.4191del , p.Argl397fs; LAMA1 c.6779C>A, p.Ser2260Tyr; LAMA2 c.3924+2T>C; LAMA2 c,1762del,p.Ala588Leufs*l l; LARGE1 exon 5-7 deletion; LEPRE1 c, 1839-2A>C; LIFR C.1258OT, p.Gln420Ter; MAN1B1 c,1376del,p.Gly459Alafs*12; METTL5 c.541+lG>C; MMUT c.810_811delins,p.Ala271Leufs*ll; MUT c.785G>T,p.Ser262Ile; NDUFS4 c.350+lG>A; NKX6-2 c,196delC,p.R66fs*122; NPC1 c.2130+lG>A; NPC1 c.3609_3610delAC, р.L1204YfsX53; NPHP3 c.3675dupT,p.Alal226fs; NPHP3 c.3674_3675insA; NRAP с.400_407del, p.Cysl34Serfs*12; OTC c.275G>A; p.Arg92Gln; PCCA c.l l l_130del,p.Tyr391Asnfs*5; PCCA c. 425G>A,p.Glyl42Asp; PGAP3 c.850C>T; р.His284Tyr; PI4KA c.3O53C>T ,p.SerlO18Leu; PKHD1 c.9374C>G, p.Ser3125*; PKHD1 с.2180A>G,p.Asn727Ser; PLA2G6 c.2070_2072del; p.Val691del; PLA2G6 c,1933C>T; р.Arg645*; POLR1C c.934T>C, p.Ser312Pro; RAB3GAP1 c,1009C>T,p.Arg337*; RAG1 с.555del,p.Lysl86Serfs*15; RAG2 c, 1332C>G, p.Ile444Met; RNASEH2C c.202C>G,p.Leu68Val; SC02 c.2T>C / p.Metl; SMN1 deletion of exons 7 and 8; SUOX c.520del;p.Aspl74Thrfs*13; SURF1 c.758_759del,p.Thr253Serfs*38; TBCE c,155_166dell2bp, p.S52_G55del; TTN c.35054del,p.Hisl l685Leufs*15; WDR62 c.758_760delACA, p.Asn253del; and WNTA c.874OT,p.Arg292Cys.

12. The method of claim 1, further comprising sequencing one or more positive control spikein fragmented DNA samples each corresponding to a 0, 1, or 2 copy number of the one or more target genes.

13. The method of claim 1, wherein the maternal genomic DNA, the cfDNA, or both are from blood or component thereof of the mother.

14. The method of claim 13, wherein the blood or component thereof is plasma.

15. A method of treating or preventing an autosomal recessive disorder or a symptom thereof in a fetus comprising: detecting a presence of an autosomal recessive disorder in the fetus by performing a method of any one of claims 1-14; and treating or preventing the autosomal recessive disorder or a symptom thereof during gestation or post-partum.

16. The method of claim 15, wherein treating or preventing comprises administering a genetic modifying agent to the fetus during gestation and / or to a neonate, child, or adult post-partum, where the genetic modifying agent is configured to modify the variant allele causative of the autosomal recessive disease or disorder such that it is an allele variant that does not result in autosomal recessive disease or disorder.

17. A kit comprising: one or more primer sets, wherein each primer set is configured to amplify a region of a target gene of an autosomal recessive variant allele and / or autosomal recessive disorder and are further configured for use in a method of any one of claims 1-14.

18. The kit of claim 16, wherein the target gene is selected from the group consisting of: SMN1, ACADVL, ADA2, ADAT3, AGL, AK2, ALDH7A1, ARSA, ASL, B3GALT6, C12orf57, Ca2, CANT1, CEP290, CTFR, CNPY3, C0G6, COL6A2, CUP2U1, DLD, EPCAM, FARS2, FKRP, GAA, GALC, GALNS, GBA, GLB1, GNPAT, GNPTG, GRM7, GUSB, HBB, HEXB, HIBCH, HSPG2, INVS, ISCA2, KIAA0586, LAMA1, LAMA2, LARGE1, LEPRE1, LIFR, MAN1B1, METTL5, MMUT, MUT, NDUFS4, NKX6-2, NPC1, NPHP3, NRAP, OTC, PCCA, PGAP3, PI4KA, PKHD1, PLA2G6, POLR1C, RAB3GAP1, RAG1, RAG2, RNASEH2C, SCO2, SUOX, SURF1, TBCE, TTN, WDR62, WNT7A, or any combination thereof.

19. The kit of claim 17, wherein the target gene is a target gene variant selected from the group consisting of: ACADVL c.65C>A,p.Ser22Ter; ACADVL c, 134C>A, p.Ser45*; ACADVL c, 1048G>A, p.Gly350Arg / c,134C>A, p.Ser45*; ACADVL c, 134C>A, p.Ser45*; ADA2 c.882- 2A>G; ADAT3 c.430G>A,p.Vall44Met; AGL c.294-2A>T; AK2 c.524G>C,p.Argl75Pro; ALDH7A1 c.518-2A>C; ARSA c,1108-2A>G; ASL c,1060C<T, p.Gln354*; B3GALT6 c.77T>C, p.Leu26Pro; C12orf57 c. lA>G,p.MetlVal; Ca2 c.232+lG>A; CANT1 c. 894_898dupGCCGC, p.Ser303Alafs*21; CANT1 c.902_906dupGCGCC, p.Ser303fs*21; CFTR c,1418del; CFTR p.Gly473Glufs*54; CNPY3 c.496-3_496-2delCA; C0G6 c.l 167-24A>G; COL6A2 c.2422+lG>A; COL6A2 C.1817-1G>A; CYP2U1 c.947A>T,p.Asp316Val; DLD c.685G>T,p.Gly229Cys; EPCAM c.583dup; EPCAM c.499dup, p.Glnl67Profs*21; EPCAM c.367G>C,p.Glyl23Arg; EPCAM c.451G>C; FARS2 c.431A>G,p.Tyrl44Cys; FKRP c.941C>T, р.Thr314Met; FKRP c, 1364C>A, p.Ala455Asp; GAA c, 1657C>T,p.Q553*; GAA с, 1657C>T,p.Q553X; GALC c,1886G>T,p.W629L; GALNS c.958G>A, p.Glu320Lys; GALNS c.970G>C, p.Ala324Pro; GBA c, 1448T>C,p.Leu483Pro; GLB1 c.622C>T,p.Arg208Cys; GNPAT c.569-3T>G; GRM7 c.461T>C ,p.Ilel54Thr; GUSB c, 1832G>A,p.Arg611Gln; HBB c.315+lG>A; HEXB c.94C>T, p.Gln32*; HIBCH c.410C>T, p.Alal37Val; HSPG2 C.1 1674+1G>T; INVS c,1807C>T, p.Arg603*; ISCA2 c.229G>A, p.Gly77Ser; KIAA0586 c.4191del , p.Argl397fs; LAMA1 C.6779OA, p.Ser2260Tyr; LAMA2 c.3924+2T>C; LAMA2 c,1762del,p.Ala588Leufs*ll; LARGE1 exon 5-7 deletion; LEPRE1 c, 1839-2A>C; LIFR c, 1258C>T, p.Gln420Ter; MAN1B1 c,1376del,p.Gly459Alafs*12; METTL5 c.541+lG>C;MMUT c.810_811delins,p.Ala271Leufs*l l; MUT c.785G>T,p.Ser262Ile; NDUFS4 c.350+lG>A; NKX6-2 c,196delC,p.R66fs*122; NPC1 c.2130+lG>A; NPC1 c.3609_3610delAC, р.L1204YfsX53; NPHP3 c.3675dupT,p.Alal226fs; NPHP3 c.3674_3675insA; NRAP с.400_407del, p.Cysl34Serfs*12; OTC c.275G>A; p.Arg92Gln; PCCA c.l ll_130del,p.Tyr391Asnfs*5; PCCA c. 425G>A,p.Glyl42Asp; PGAP3 c.850C>T; р.His284Tyr; PI4KA c.3O53C>T ,p.SerlO18Leu; PKHD1 c.9374C>G, p.Ser3125*; PKHD1 с.2180A>G,p.Asn727Ser; PLA2G6 c.2070_2072del; p.Val691del; PLA2G6 c,1933C>T; р.Arg645*; POLR1C c.934T>C, p.Ser312Pro; RAB3GAP1 c,1009C>T,p.Arg337*; RAG1 с.555del,p.Lysl86Serfs*15; RAG2 C.1332OG, p.Ile444Met; RNASEH2C c.202C>G,p.Leu68Val; SCO2 c.2T>C / p.Metl; SMN1 deletion of exons 7 and 8; SUOXc.520del; p.Aspl74Thrfs*13; SURF1 c.758 759del,p.Thr253Serfs*38; TBCE c.155 166dell2bp, p.S52_G55del; TTN c.35054del,p.Hisl 1685Leufs*15; WDR62 c.758_760delACA, p.Asn253del; and WNTA c.874C>T,p.Arg292Cys, or any combination thereof.

20. The kit of claim 17, further comprising one or more reagents for carrying out polymerase chain reaction, sequencing, or both.

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