Methods for determination and treatment of rhesus incompatibilty

Abstract

Disclosed herein includes a method of determining the RhD genotype of a fetus, comprising: (a) extracting cell-free DNA from a sample of a pregnant person, wherein the extracted DNA comprises a mixture of maternal cell-free DNA and fetal cell-free DNA; (b) performing targeted multiplex amplification on the extracted DNA to amplify a plurality of target loci together in the same reaction mixture using a plurality of primer pairs, wherein at least three of the primer pairs each targets a different exon or intron of the RhD gene and the RhCE homologue gene and is each designed to amplify a target locus that comprises a variant between the RhD gene and the RhCE homologue gene; and (c) sequencing the amplicons by high-throughput sequencing and determine the RhD genotype of the fetus using the sequencing reads of the amplicons targeting the RhD gene and the RhCE homologue gene.

Description

METHODS FOR DETERMINATION AND TREATMENT OF RHESUS INCOMPATIBILTYCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefits of U.S. Provisional Application No. 63 / 666,019 filed June 28, 2024, the contents of which are hereby incorporated by reference in their entirety.BACKGROUND

[0002] Rhesus (Rh) incompatibility refers to the discordant pairing of maternal and fetal Rh types. It is associated with the development of maternal Rh sensitization and hemolytic disease of the neonate (HDN). An individual can be classified as RhD positive if their erythrocytes express the RhD antigen; otherwise, an individual is RhD negative if they do not. This phenomenon becomes clinically significant if a mother that is RhD negative becomes sensitized to the D antigen and subsequently, produces anti-D antibodies (i.e., alloimmunization) that can bind to and potentially lead to the destruction of RhD positive erythrocytes. This is of particular concern if an RhD negative mother is carrying an RhD positive fetus, which can result in consequences along the spectrum of HDN ranging from self-limited hemolytic anemia to severe hydrops fetalis.

[0003] Currently, management of Rh incompatibility in RhD negative people, regardless of the Rh status of the fetus, includes administration of prophylactic RhD immunoglobulin (Rhlg) at 28 weeks gestation. If the neonate is found to be Rh-positive after delivery, those same unsensitized Rh- negative people should be given Rhlg within 72 hours of delivery. This method however results in unnecessary treatment of pregnant people carrying Rh negative fetuses.

[0004] Therefore, there remains a need for an accurate and non-invasive test to determine Rh incompatibility between a mother and fetus, to avoid unnecessary treatment of the mother.SUMMARY

[0005] In one aspect, the present disclosure provides a method of determining the RhD genotype of a fetus, comprising: (a) extracting cell-free DNA from a blood, plasma, or serum sample of a pregnant person, wherein the extracted DNA comprises a mixture of maternal cell-free DNA andfetal cell-free DNA; (b) performing targeted multiplex amplification on the extracted DNA to amplify a plurality of target loci together in the same reaction mixture using a plurality of primer pairs, wherein at least three of the primer pairs each targets a different exon or intron of the RhD and RhCE genes and is each designed to amplify a target locus that comprises a variant between the RhD gene and the RhCE homologue gene; (c) sequencing the amplicons by high-throughput sequencing to generate sequencing reads of the target loci and determine the RhD genotype of the fetus.

[0006] In some embodiments, the pregnant person is RhD negative.

[0007] In some embodiments, at least five of the primer pairs each targets a different exon or intron of the RhD and RhCE genes and is each designed to amplify a target locus that comprises a variant between the RhD gene and the RhCE homologue gene.

[0008] In some embodiments, at least ten of the primer pairs each targets a different exon or intron of the RhD and RhCE genes and is each designed to amplify a target locus that comprises a variant between the RhD gene and the RhCE homologue gene.

[0009] In some embodiments, a target locus in exon 7 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene is amplified and sequenced.

[0010] In some embodiments, a target locus in exon 9 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene is amplified and sequenced.

[0011] In some embodiments, a target locus in exon 6 of the RhD and RhCE genes that comprises a nonsense variant between the RhD gene and the RhD pseudogene is amplified and sequenced.

[0012] In some embodiments, the targeted multiplex amplification comprises amplification of at least 100, at least 500, at least 1,000, or at least 10,000 target loci together in the same reaction mixture.

[0013] In some embodiments, the targeted multiplex amplification comprises amplification of 100 to 20,000 target loci on one or more chromosomes expected to be disomic.

[0014] In some embodiments, step (a) further comprises size selection of the cell-free DNA.

[0015] In some embodiments, the primer pairs are each designed to amplify less than about 100 bp of DNA, less than about 80 bp of DNA, or about 65-80 by of DNA.

[0016] In some embodiments, the target loci comprise single nucleotide polymorphism and / or single nucleotide variant loci.

[0017] In some embodiments, step (c) comprises performing a barcoding PCR to add a sequencing tag and a sample- specific barcode to obtain a barcoded sequencing library, pooling a plurality of barcoded sequencing libraries and sequencing the pool of barcoded sequencing libraries together in one sequencing lane.

[0018] In some embodiments, both RhD and RhCE sequencing reads are present, and the RhD genotype of the fetus is RhD+.

[0019] In some embodiments, only RhCE sequencing reads are present, and the RhD genotype of the fetus is RhD deletion.

[0020] In some embodiments, only RhCE sequencing reads are present for exons 4-7 of the RhD gene, and the RhD genotype of the fetus is RhD-CE-D hybrid.

[0021] In some embodiments, sequencing reads corresponding to a duplication insert and / or a nucleotide change that creates a stop codon are present, and the RhD genotype of the fetus is RhD pseudogene.

[0022] In some embodiments, the pregnant person is at a gestational age of at least 9 weeks or between 9 and 20 weeks.

[0023] In another aspect, the present disclosure provides a method of determining Rhesus incompatibility, comprising: (a) genotyping a pregnant person to determine the RhD genotype of the pregnant person; (b) extracting cell-free DNA from a blood, plasma, or serum sample of thepregnant person who is RhD negative, wherein the extracted DNA comprises a mixture of maternal cell-free DNA and fetal cell-free DNA; (c) performing targeted multiplex amplification on the extracted DNA to amplify a plurality of target loci together in the same reaction mixture using a plurality of primer pairs, wherein at least three of the primer pairs each targets a different exon or intron of the RhD and RhCE genes and is each designed to amplify a target locus that comprises a variant between the RhD gene and the RhCE homologue gene; (d) sequencing the amplicons by high-throughput sequencing to generate sequencing reads of the target loci and determine the RhD genotype of the fetus and the presence or absence of Rhesus incompatibility between the pregnant person and the fetus.

[0024] In a further aspect, the present disclosure provides a method of treatment of Rhesus incompatibility, comprising: (a) extracting cell-free DNA from a blood, plasma, or serum sample of a pregnant person who is RhD negative, wherein the extracted DNA comprises a mixture of maternal cell-free DNA and fetal cell-free DNA; (b) performing targeted multiplex amplification on the extracted DNA to amplify a plurality of target loci together in the same reaction mixture using a plurality of primer pairs, wherein at least three of the primer pairs each targets a different exon or intron of the RhD and RhCE genes and is each designed to amplify a target locus that comprises a variant between the RhD gene and the RhCE homologue gene; (c) sequencing the amplicons by high-throughput sequencing to generate sequencing reads of the target loci and determine the RhD genotype of the fetus and the presence or absence of Rhesus incompatibility between the pregnant person and the fetus; and (d) administering Rh immune-globulin to the pregnant person determined to have Rhesus incompatibility between the pregnant person and the fetus.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1. The RhD / RhCE locus is present on chromosome 1, and the RhD negative phenotype is found in approximately 15% of the population. Compared to the RhD positive phenotype (FIG. 1 A), a majority of RhD negative phenotypes can be caused by one of three main mechanisms: 1. RhD deletion, where the entire RhD gene is deleted and a hybrid Rh box containing portions of two terminal Rh boxes is present (FIG. IB); 2. RhD*psi (or RhD*'P), a pseudogene with a duplication insert and a nucleotide change that creates a stop codon (FIG.1C); and 3. RhD-CE-D hybrid (or RhD*IIIa-CE(4-7)-D), in which certain RhD exons are deleted and replaced by RhCE exons (FIG. ID.). A set of primers for targeted amplicons covering regions differing between the RhD and RhCE genes were designed and incorporated into a SNP- based NIPT assay. RhD and RhCE can be directly detected by targeting the differences between these two highly similar genes.

[0026] Figure 2. Assay Performance for cfDNA fetal RhD Test. The analysis included 655 Rh negative pregnant people who had previous prenatal cfDNA screening for fetal aneuploidy had documented fetal or newborn Rh blood type (serological truth).DETAILED DESCRIPTION

[0027] The present invention generally relates to the field of non-invasive prenatal screening (NIPS), particularly NIPS using cell-free DNA (cfDNA) found in maternal plasma. More particularly, the present invention provides methods, compositions and kits suitable for identifying fetal Rh status in maternal blood samples of Rh negative pregnant patients. More particularly, the present invention provides methods, compositions and kits suitable for identifying Rh incompatibility in maternal blood samples. The invention is based on amplification and detection of RhD and RhCE amplicons.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the present technology belongs.

[0029] As used herein, unless otherwise stated, the singular forms “a,” “an,” and “the” include plural reference. Thus, for example, a reference to “an oligonucleotide” includes a plurality of oligonucleotide molecules, and a reference to “a nucleic acid” is a reference to one or more nucleic acids.

[0030] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1 %— 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context.

[0031] Extraction refers to any action taken to remove nucleic acids from other (non-nucleic acid) material present in the sample. The term extraction includes mechanical or chemical lysis, addition of detergent or protease, or precipitation and removal of non-nucleic acids such as proteins.

[0032] Sample refers to clinical samples obtained from a patient. In preferred embodiments, a sample is obtained from a biological source (z.e., a “biological sample”), such as tissue, bodily fluid, or microorganisms collected from a subject. Sample sources include, but are not limited to, mucus, sputum (processed or unprocessed), bronchial alveolar' lavage (BAL), bronchial wash (BW), blood, bodily fluids, cerebrospinal fluid (CSF), urine, plasma, serum, or tissue (e.g., biopsy material).

[0033] Target nucleic acid or “target sequence” refers to a nucleic acid sequence of interest to be detected and / or quantified in the sample to be analyzed. Target nucleic acid may be composed of segments of a chromosome, a complete gene with or without intergenic sequence, segments or portions of a gene with or without intergenic sequence, or sequence of nucleic acids which probes or primers are designed. Target nucleic acids may include a wild-type sequence(s), a mutation, deletion, insertion or duplication, tandem repeat elements, a gene of interest, a region of a gene of interest or any upstream or downstream region thereof. Target nucleic acids may represent alternative sequences or alleles of a particular gene. Target nucleic acids may be derived from genomic DNA, cDNA, or RNA.

[0034] Single Nucleotide Polymorphism (SNP) refers to a single nucleotide that may differ between the genomes of two members of the same species. The usage of the term should not imply any limit on the frequency with which each variant occurs.

[0035] Nucleic acid refers to a deoxyribonucleotide (DNA), ribonucleotide polymer (RNA), RNA / DNA hybrids and polyamide nucleic acids (PNAs) in either single- or double- stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.

[0036] Locus refers to a particular region of interest on the DNA (or corresponding RNA) of an individual, which may refer to a SNP, the site of a possible insertion or deletion, or the site of some other relevant genetic variation. Disease-linked SNPs may also refer to disease-linked loci.

[0037] Polymorphic Allele, also “Polymorphic Locus,” refers to an allele or locus where the genotype varies between individuals within a given species. Some examples of polymorphic alleles include single nucleotide polymorphisms, short tandem repeats, deletions, duplications, and inversions.

[0038] Allele refers to the alternative form or version of a gene that occupies a particular locus. Genetic Data also '"Genotypic Data ” refers to the data describing aspects of the genome of one or more individuals. It may refer to one or a set of loci, partial or entire sequences, partial or entire chromosomes, or the entire genome. It may refer to the identity of one or a plurality of nucleotides; it may refer to a set of sequential nucleotides, or nucleotides from different locations in the genome, or a combination thereof. Genotypic data is typically in silica, however, it is also possible to consider physical nucleotides in a sequence as chemically encoded genetic data. Genotypic Data may be said to be “on,” “of,” “at,” “from” or “on” the individual(s). Genotypic Data may refer to output measurements from a genotyping platform where those measurements are made on genetic material.

[0039] Genetic Material also "Genetic Sample’’ refers to physical matter, such as tissue or blood, from one or more individuals comprising DNA or RNA

[0040] Confidence refers to the statistical likelihood that the called SNP, allele, set of alleles, ploidy call, or determined number of chromosome segment copies correctly represents the real genetic state of the individual.

[0041] Chromosome may refer to a single chromosome copy, meaning a single molecule of DNA of which there are 46 in a normal somatic cell; an example is ‘the maternally derived chromosome 18’. Chromosome may also refer to a chromosome type, of which there are 23 in a normal human somatic cell; an example is ‘chromosome 18’.

[0042] Chromosomal Identity may refer to the referent chromosome number, i.e. the chromosome type. Normal humans have 22 types of numbered autosomal chromosome types, and two types of sex chromosomes. It may also refer to the parental origin of the chromosome. It may also refer to a specific chromosome inherited from the parent. It may also refer to other identifying features of a chromosome.

[0043] Allelic Data refers to a set of genotypic data concerning a set of one or more alleles. It may refer to the phased, haplotypic data. It may refer to SNP identities, and it may refer to the sequence data of the DNA, including insertions, deletions, repeats and mutations. It may include the parental origin of each allele.

[0044] Allelic State refers to the actual state of the genes in a set of one or more alleles. It may refer to the actual state of the genes described by the allelic data.

[0045] Allelic Ratio or allele ratio, refers to the ratio between the amount of each allele at a locus that is present in a sample or in an individual. When the sample was measured by sequencing, the allelic ratio may refer to the ratio of sequence reads that map to each allele at the locus. When the sample was measured by an intensity based measurement method, the allele ratio may refer to the ratio of the amounts of each allele present at that locus as estimated by the measurement method.

[0046] Allele Count refers to the number of sequences that map to a particular locus, and if that locus is polymorphic, it refers to the number of sequences that map to each of the alleles. If each allele is counted in a binary fashion, then the allele count will be whole number. If the alleles are counted probabilistically, then the allele count can be a fractional number.

[0047] Allele Count Probability refers to the number of sequences that are likely to map to a particular locus or a set of alleles at a polymorphic locus, combined with the probability of the mapping. Note that allele counts are equivalent to allele count probabilities where the probability of the mapping for each counted sequence is binary (zero or one). In some embodiments, the allele count probabilities may be binary. In some embodiments, the allele count probabilities may be set to be equal to the DNA measurements.

[0048] Allelic Distribution, or ‘allele count distribution’ refers to the relative amount of each allele that is present for each locus in a set of loci. An allelic distribution can refer to an individual, to a sample, or to a set of measurements made on a sample. In the context of sequencing, the allelic distribution refers to the number or probable number of reads that map to a particular allele for each allele in a set of polymorphic loci. The allele measurements may be treated probabilistically, that is, the likelihood that a given allele is present for a give sequence read is a fraction between 0and 1 , or they may be treated in a binary fashion, that is, any given read is considered to be exactly zero or one copies of a particular allele.

[0049] Allelic Distribution Pattern refers to a set of different allele distributions for different parental contexts. Certain allelic distribution patterns may be indicative of certain ploidy states.

[0050] Allelic Bias refers to the degree to which the measured ratio of alleles at a heterozygous locus is different to the ratio that was present in the original sample, such as a sample of DNA. The degree of allelic bias at a particular locus is equal to the observed allelic ratio at that locus, as measured, divided by the ratio of alleles in the original DNA or RNA sample at that locus. Allelic bias may be defined to be greater than one, such that if the calculation of the degree of allelic bias returns a value, x, that is less than 1, then the degree of allelic bias may be restated as 1 / x. Allelic bias maybe due to amplification bias, purification bias, or some other phenomenon that affects different alleles differently.

[0051] Allelic imbalance for aneuploidy determinations, such as CNV determinations, refers to the difference between the frequencies of the alleles for a locus. It is an estimate of the difference in the copy of numbers of the homologs. Allelic imbalance can arise from the complete loss of an allele or from an increase in copy number of one allele relative to the other. Allelic imbalances can be detected by measuring the proportion of one allele relative to the other in fluids or cells from individuals that arc constitutionally heterozygous at a given locus. (Mei ct al, Genome Res, 10:1126-37 (2000)). For dimorphic SNPs that have alleles arbitrarily designated ‘A’ and ‘B’, the allele ratio of the A allele is nA / (nA + ns), where nA and ns are the number of sequencing reads for alleles A and B, respectively. Allelic imbalance is the difference between the allele ratios of A and B for loci that arc heterozygous in the germline. This definition is analogous to that for SNVs, where the proportion of abnormal DNA is typically measured using mutant allele frequency, or nm / (nm+ nr), where nmand nrare the number of sequencing reads for the mutant allele and the reference allele, respectively. Accordingly, the proportion of abnormal DNA for a CNV can be measured by the average allelic imbalance (AAI), defined as l(H 1 - H2)I / (H1 + H2), where Hi is the average number of copies of homolog i in the sample and Hi / (H1 + H2) is the fractional abundance, or homolog ratio, of homolog i. The maximum homolog ratio is the homolog ratio of the more abundant homolog.

[0052] Cell-free DNA or “cfDNA” refers to any free-floating DNA existing in a sample, such as the blood plasma of a pregnant patient. Cell-free DNA found in a pregnant person's blood may contain DNA originating from both the mother and the fetus. The term “cell-free fetal DNA” or “cffDNA” as used herein refers to fetal DNA circulating freely in the maternal system, such as in the mother's bloodstream. Through various mechanisms, cffDNA may, for example, originate from the trophoblasts making up the placenta. In some cases, the fetal DNA may be fragmented and make its way into the maternal bloodstream via shedding of the placental micro-particles into the maternal bloodstream. In some cases, cflDNA can first be observed in maternal blood as early as 7 weeks gestation, and increases in the amount as the pregnancy progresses. The cffDNA may be sampled by venipuncture on the mother and provides the basis for non-invasive prenatal diagnosis and testing.

[0053] Fetal fraction refers to the percentage of cell-free DNA found in a pregnant mother's test sample that originates from the fetus. For example, if 10% of cell-free DNA found in a mother's blood sample is of a fetal origin, the fetal fraction is determined to be 10%. In some embodiments, fetal fraction is used as a parameter for sample quality and for determining whether a maternal sample should be included in the analysis. Particularly, in some embodiments, when the fetal fraction of a sample is below a predetermined threshold, the maternal sample is excluded. In some embodiments, the threshold ranges between about 1% to about 5%. In some embodiments, the threshold is about 4%.

[0054] Library refers to a collection of nucleic acid sequences, e. ., a collection of nucleic acids derived from whole genomic, sub-genomic fragments, cell-free DNA, cell-free DNA fragments, cDNA, cDNA fragments, RNA, RNA fragments, or a combination thereof. In one embodiment, a portion or all of the library nucleic acid sequences comprises an adapter sequence. The adapter sequence can be located at one or both ends. The adapter sequence can be useful, e.g., for a sequencing method (e.g., an NGS method), for amplification, for reverse transcription, or for cloning into a vector. In some embodiments described here, the libraries are constructed from cell-fee DNA and / or gDNA.

[0055] Oligonucleotide refers to a molecule that has a sequence of nucleic acid bases on a backbone comprised mainly of identical monomer units at defined intervals. The bases arearranged on the backbone in such a way that they can bind with a nucleic acid having a sequence of bases that arc complementary to the bases of the oligonucleotide. The most common oligonucleotides have a backbone of sugar phosphate units. A distinction may be made between oligodeoxyribonucleotides that do not have a hydroxyl group at the 2' position and oligoribonucleotides that have a hydroxyl group at the 2' position. Oligonucleotides may also include derivatives, in which the hydrogen of the hydroxyl group is replaced with organic groups, e.g., an allyl group. Oligonucleotides that function as primers or probes are generally at least about 10-15 nucleotides in length or up to about 70, 100, 110, 150 or 200 nucleotides in length, and more preferably at least about 15 to 25 nucleotides in length. Oligonucleotides used as primers or probes for specifically amplifying or specifically detecting a particular target nucleic acid generally are capable of specifically hybridizing to the target nucleic acid.

[0056] A positive control nucleic acid or “internal positive amplification control” refers to a nucleic acid known to be present in a sample at a certain amount or level. In some embodiments, a positive control nucleic acid is not naturally present in a sample and is added to the sample prior to subjecting the reaction-sample mixture to real-time polymerase chain reaction. In some embodiments, the positive control nucleic acid is an artificially prepared DNA sample comprising a percentage of RhD positive DNA. In some embodiments, the positive control nucleic acid comprises between 0.1% and 100% RhD positive DNA.

[0057] Primer, also “PCR probe” refers to a single DNA molecule (a DNA oligomer) or a collection of DNA molecules (DNA oligomers) where the DNA molecules are identical, or nearly so, and where the primer contains a region that is designed to hybridize to a targeted locus e.g, a targeted polymorphic locus or a nonpolymorphic locus), and may contain a priming sequence designed to allow PCR amplification. A primer may also contain a molecular barcode. A primer may contain a random region that differs for each individual molecule. The terms “test primer” and “candidate primer” are not meant to be limiting and may refer to any of the primers disclosed herein.

[0058] Library of primers refers to a population of two or more primers. In various embodiments, the library includes at least 25; 50; 75; 100; 300; 500; 750; 1,000; 2,000; 5,000; 7,500; 10,000; 15,000; 19,000; 20,000; 25,000; 27,000; 28,000; 30,000; 40,000; 50,000; 75,000; or 100,000different primers. In various embodiments, the library includes at least 25; 50; 75; 100; 300; 500; 750; 1,000; 2,000; 5,000; 7,500; 10,000; 15,000; 19,000; 20,000; 25,000; 27,000; 28,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different primer pairs, wherein each pair of primers includes a forward test primer and a reverse test primer where each pair of test primers hybridize to a target locus. In some embodiments, the library of primers includes at least 25; 50; 75; 100; 300; 500; 750; 1,000; 2,000; 5,000; 7,500; 10,000; 15,000; 19,000; 20,000; 25,000; 27,000; 28,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different individual primers that each hybridize to a different target locus, wherein the individual primers are not pail of primer pairs. In some embodiments, the library has both (i) primer pairs and (ii) individual primers (such as universal primers) that are not part of primer pairs. Primers are typically at least 10, 15, 18, or 30 nucleotides in length or up to about 100, 110, 125, or 200 nucleotides in length. In some embodiments, primers are preferably between about 15 to about 60 nucleotides in length, and most preferably between about 25 to about 40 nucleotides in length. In some embodiments, primers are 15 to 35 nucleotides in length. There is no standard length for optimal hybridization or polymerase chain reaction amplification.

[0059] Hybrid Capture Probe refers to any nucleic acid sequence, possibly modified, that is generated by various methods such as PCR or direct synthesis and intended to be complementary to one strand of a specific target DNA sequence in a sample. The exogenous hybrid capture probes may be added to a prepared sample and hybridized through a denature-reannealing process to form duplexes of exogenous-endogenous fragments. These duplexes may then be physically separated from the sample by various means.

[0060] Hybridize refers to a process where two substantially complementary nucleic acid strands (at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, at least about 75%, or at least about 90% complementary) anneal to each other under appropriately stringent conditions to form a duplex or heteroduplex through formation of hydrogen bonds between complementary base pairs. Hybridizations are typically and preferably conducted with probelength nucleic acid molecules, preferably 15-100 nucleotides in length, more preferably 18-50 nucleotides in length. Nucleic acid hybridization techniques are well known in the art. See, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y. Hybridization and the strength of hybridization (z.e., the strength of the association between the nucleic acids) is influenced by such factors as the degree ofcomplementarity between the nucleic acids, stringency of the conditions involved, and the thermal melting point (Tm) of the formed hybrid. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions such that sequences having at least a desired level of complementarity will stably hybridize, while those having lower complementarity will not. For examples of hybridization conditions and parameters, see, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y.; Ausubel, F. M. etal. 1994, Current Protocols in Molecular Biology, John Wiley & Sons, Secaucus, N.J. In some embodiments, specific hybridization occurs under stringent hybridization conditions. An oligonucleotide or polynucleotide (e.g., a probe or a primer) that is specific for a target nucleic acid will “hybridize” to the target nucleic acid under suitable conditions.

[0061] Next generation sequencing or “NGS” refers to any sequencing method that determines the nucleotide sequence of either individual nucleic acid molecules (e.g., in single molecule sequencing) or clonally expanded proxies for individual nucleic acid molecules in a high throughput parallel fashion (e.g., greater than 103, 104, 105or more molecules are sequenced simultaneously). In one embodiment, the relative abundance of the nucleic acid species in the library can be estimated by counting the relative number of occurrences of their cognate sequences in the data generated by the sequencing experiment. Next generation sequencing methods are known in the ail, and are described, e.g., in Metzker, M. Nature Biotechnology Reviews 11 :31-46 (2010).

[0062] Sequence read or simply “read” refers to sequence information of a nucleic acid fragment obtained through a sequencing assay, such as a next generation sequencing (NGS) assay. In some embodiments, a sequence read refers to data representing a sequence of nucleotide bases that were measured using a clonal sequencing method. Clonal sequencing may produce sequence data representing single, or clones, or clusters of one original DNA molecule. A sequence read may also have associated quality score at each base position of the sequence indicating the probability that nucleotide has been called correctly.

[0063] Mapping a sequence read is the process of determining a sequence read’s location of origin in the genome sequence of a particular organism. The location of origin of sequence reads is based on similarity of nucleotide sequence of the read and the genome sequence.

[0064] Haplotype refers to a combination of alleles at multiple loci that are typically inherited together on the same chromosome. Haplotype may refer to as few as two loci or to an entire chromosome depending on the number of recombination events that have occurred between a given set of loci. Haplotype can also refer to a set of single nucleotide polymorphisms (SNPs) on a single chromatid that are statistically associated.

[0065] Haplotypic Data, also “Phased Data” or “Ordered Genetic Data,” refers to data from a single chromosome in a diploid or polyploid genome, i.e., either the segregated maternal or paternal copy of a chromosome in a diploid genome.

[0066] Phasing refers to the act of determining the haplotypic genetic data of an individual given unordered, diploid (or polyploidy) genetic data. It may refer to the act of determining which of two genes at an allele, for a set of alleles found on one chromosome, are associated with each of the two homologous chromosomes in an individual.

[0067] Phased Data refers to genetic data where one or more haplotypes have been determined.

[0068] Hypothesis refers to a possible ploidy state at a given set of chromosomes, or a set of possible allelic states at a given set of loci. The set of possibilities may comprise one or more elements.

[0069] Copy Number Hypothesis, also “Ploidy State Hypothesis,” refers to a hypothesis concerning the number of copies of a chromosome in an individual. It may also refer to a hypothesis concerning the identity of each of the chromosomes, including the parent of origin of each chromosome, and which of the parent’s two chromosomes are present in the individual. It may also refer to a hypothesis concerning which chromosomes, or chromosome segments, if any, from a related individual correspond genetically to a given chromosome from an individual.

[0070] Fetal refers to “of the fetus,” or “of the region of the placenta that is genetically similar to the fetus”. In a pregnant person, some portion of the placenta is genetically similar to the fetus, and the free floating fetal DNA found in maternal blood may have originated from the portion of the placenta with a genotype that matches the fetus. Note that the genetic information in half of the chromosomes in a fetus is inherited from the mother of the fetus. In some embodiments, theDNA from these maternally inherited chromosomes that came from a fetal cell is considered to be “of fetal origin,” not “of maternal origin.”

[0071] DNA of Fetal Origin refers to DNA that was originally part of a cell whose genotype was essentially equivalent to that of the fetus.

[0072] DNA of Maternal Origin refers to DNA that was originally part of a cell whose genotype was essentially equivalent to that of the mother.

[0073] Non-Invasive Prenatal Diagnosis (NIPD), also “Non-Invasive Prenatal Screening” (NIPS), also “Non-invasive prenatal testing” (NIPT), or also prenatal cell-free DNA screening, refers to a method of determining the genetic state of a fetus that is gestating in a mother using genetic material found in the mother’s blood, where the genetic material is obtained by drawing the mother’s intravenous blood. In embodiments described herein, the methods diagnose, screen, or test for the fetal RhD genotype.

[0074] Preferential Enrichment of DNA that corresponds to a locus, or preferential enrichment of DNA at a locus, refers to any method that results in the percentage of molecules of DNA in a postenrichment DNA mixture that correspond to the locus being higher than the percentage of molecules of DNA in the pre-enrichment DNA mixture that correspond to the locus. The method may involve selective amplification of DNA molecules that correspond to a locus. The method may involve removing DNA molecules that do not correspond to the locus. The method may involve a combination of methods. The degree of enrichment is defined as the percentage of molecules of DNA in the post-enrichment mixture that correspond to the locus divided by the percentage of molecules of DNA in the pre-enrichment mixture that correspond to the locus. Preferential enrichment may be carried out at a plurality of loci. In some embodiments of the present disclosure, the degree of enrichment is greater than 20. In some embodiments of the present disclosure, the degree of enrichment is greater than 200. In some embodiments of the present disclosure, the degree of enrichment is greater than 2,000. When preferential enrichment is carried out at a plurality of loci, the degree of enrichment may refer to the average degree of enrichment of all of the loci in the set of loci.

[0075] Amplification, with respect to nucleic acid sequences, refer to methods that increase the representation of a population of nucleic acid sequences in a sample. Copies of a particular target nucleic acid sequence generated in vitro in an amplification reaction are called “amplicons” or “amplification products”. Amplification may be exponential or linear. A target nucleic acid may be DNA (such as, for example, genomic DNA and cDNA) or RNA. While the exemplary methods described hereinafter relate to amplification using polymerase chain reaction (PCR), numerous other methods such as isothermal methods, rolling circle methods, etc., are available to the skilled artisan. The skilled artisan will understand that these other methods may be used either in place of, or together with, PCR methods. See, e.g., Saiki, “Amplification of Genomic DNA” in PCR PROTOCOLS, Innis et al., Eds., Academic Press, San Diego, CA 1990, pp 13-20; Wharam, et al., Nucleic Acids Res. 29(11):E54-E54 (2001).

[0076] Selective Amplification may refer to a method that increases the number of copies of a particular molecule of DNA, or molecules of DNA that correspond to a particular region of DNA. It may also refer to a method that increases the number of copies of a particular targeted molecule of DNA, or targeted region of DNA more than it increases non-targeted molecules or regions of DNA. Selective amplification may be a method of preferential enrichment.

[0077] Multiplex PCR refers to the simultaneous generation of two or more PCR products or amplicons within the same reaction vessel. Similarly, a “2-plex PCR” refers to the simultaneous generation of two PCR products or amplicons within the same reaction vessel. Each PCR product is primed using a distinct primer pair. A multiplex reaction may further include specific probes for each product that are labeled with different detectable moieties.

[0078] Universal Priming Sequence refers to a DNA sequence that may be appended to a population of target DNA molecules, for example by ligation, PCR, or ligation mediated PCR. Once added to the population of target molecules, primers specific to the universal priming sequences can be used to amplify the target population using a single pair of amplification primers. Universal priming sequences are typically not related to the target sequences.

[0079] Universal Adapters, or ‘ligation adaptors’ or ‘library tags’ are DNA molecules containing a universal priming sequence that can be covalently linked to the 5-prime and 3-prime end of a population of target double stranded DNA molecules. The addition of the adapters providesuniversal priming sequences to the 5-prime and 3-prime end of the target population from which PCR amplification can take place, amplifying all molecules from the target population, using a single pair of amplification primers.

[0080] Joint Distribution Model refers to a model that defines the probability of events defined in terms of multiple random variables, given a plurality of random variables defined on the same probability space, where the probabilities of the variable are linked. In some embodiments, the degenerate case where the probabilities of the variables are not linked may be used.

[0081] RhD phenotype refers to determining the presence or absence of antigens of the Rh blood group, specifically red cell antigen D. An individual is either RhD positive or RhD negative for a given antigen. For example, a RhD negative individual does not express antigen D, whereas an RhD positive individual does express antigen D.

[0082] Rh negative refers to a RhD negative phenotype. The RhD negative phenotype, found in approximately 15% of the population, can be caused by three main mechanisms: 1. RhD deletion, where the entire RhD gene is deleted (FIG. IB); 2. RhD*psi (or RhD* ), a pseudo gene with a duplication insert and a nucleotide change that creates a stop codon (FIG. 1C); and 3. RhD-CE-D hybrid (or RhD*IIIa-CE(4-7)-D), in which certain RhD exons arc deleted and replaced by RHCE exons (FIG. ID.).

[0083] RhD incompatibility occurs when an RhD negative mother is pregnant with an RhD positive fetus. This phenomenon becomes clinically significant if a mother that is RhD negative becomes sensitized to the D antigen and subsequently produces anti-D antibodies (i.e., alloimmunization) that can bind to and potentially lead to the destruction of RhD positive erythrocytes. This is of particular concern if an RhD negative mother is carrying an RhD positive fetus, which can result in consequences along the spectrum of HDN ranging from self-limited hemolytic anemia to severe hydrops fetalis.

[0084] Maternal RhD sensitization occurs when red cells from a RhD positive fetus cross the placenta and sensitize an RhD negative mother, especially at parturition. When mother that is RhD negative becomes sensitized to the Rh antigens present on the red blood cells, the mother willproduce anti-Rh antibodies (i.e., alloimmunization). The mother's antibodies may then, in a subsequent pregnancy, cause haemolytic disease of the newborn if the fetus is Rhesus positive.Rhesus Incompatibility

[0085] Rh incompatibility between a pregnant mother and her in utero fetus exists when the mother has Rh negative blood and her fetus has Rh positive blood (e.g., due to the fetus's father having Rh positive blood). During pregnancy, when blood from an Rh positive fetus mixes with blood from an Rh negative mother, the mother's body naturally generates antibodies that may affect the current pregnancy with the fetus and / or future pregnancies with other fetus’, in such examples, the antibodies may target and / or attack Rh proteins in the blood of the fetus and / or in the blood of any babies of future pregnancies. Such attacks may cause a decrease in red blood cells and potentially cause a plurality of diseases and / or complications (e.g., anemia, jaundice, brain damage, heart failure, or death for the fetus(s).

[0086] When an RhD incompatibility exists between a mother and her fetus, treatment is available for the mother to prevent generation of antibodies (e.g., by administering injections of Rh immunoglobulin) and / or destruction of any antibodies that have been generated. Accordingly, the treatment may maintain safety of the fetus by protecting the fetus's RhD positive blood and or red blood cells. Previous techniques for detecting Rh incompatibility between an RhD negative mother and her RhD positive fetus involve various invasive methods (e.g., amniocentesis, including probing the uterus of the mother) and non-invasive methods (e.g., ultrasounds, maternal blood sampling). Invasive methods may be painful and / or, in some instances, pose risk to the mother and / or fetus. Previous non-invasive methods, though considered relatively safe, are performed periodically (e.g., monthly) by a mother's healthcare provider. Accordingly, a considerable amount of time (up to a month or more) may pass between an existence of Rh incompatibility taking place and detection of the Rh incompatibility. In some instances, the mother’s antibodies may already have caused untreatable damage to the fetus and / or the fetus's blood system. Thus, it is desirable to detect an Rh incompatibility between a mother and her fetus as soon as possible so that the mother may receive treatment to ensure the safety of the fetus.Methods for Determination of Rhesus Incompatibility

[0087] In one aspect, the present disclosure provides a method of determining the RhD genotype of a fetus, comprising: (a) extracting ccll-frcc DNA from a blood, plasma, or scrum sample of a pregnant person, wherein the extracted DNA comprises a mixture of maternal cell-free DNA and fetal cell-free DNA; (b) performing targeted multiplex amplification on the extracted DNA to amplify a plurality of target loci together in the same reaction mixture using a plurality of primer pairs, wherein at least three of the primer pairs each targets a different exon or intron of the RhD and RhCE genes and is each designed to amplify a target locus that comprises a variant between the RhD gene and the RhCE homologue gene; (c) sequencing the amplicons by high-throughput sequencing to generate sequencing reads of the target loci and determine the RhD genotype of the fetus.

[0088] In some embodiments, the pregnant person is RhD negative. In some embodiments, the pregnant person has been determined to be RhD negative by performing a blood type and screen (serology). Accordingly, in another aspect, the present disclosure provides a method of determining Rhesus incompatibility, comprising: (a) performing a blood type and screen on the pregnant person, or genotyping a pregnant person, to determine the RhD genotype of the pregnant person; (b) extracting cell-free DNA from a blood, plasma, or serum sample of the pregnant person who is RhD negative, wherein the extracted DNA comprises a mixture of maternal cell-free DNA and fetal cell-free DNA; (c) performing targeted multiplex amplification on the extracted DNA to amplify a plurality of target loci together in the same reaction mixture using a plurality of primer pairs, wherein at least three of the primer pairs each targets a different exon or intron of the RhD and RhCE genes and is each designed to amplify a target locus that comprises a variant between the RhD gene and the RhCE homologue gene; (d) sequencing the amplicons by high-throughput sequencing to generate sequencing reads of the target loci and determine the RhD genotype of the fetus and the presence or absence of Rhesus incompatibility between the pregnant person and the fetus.

[0089] In some embodiments, the targeted multiplex amplification comprises amplification of at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 target loci together in the same reaction mixture. In some embodiments, the targeted multiplex amplification comprises amplification of no more than 20,000, no more than 10,000, no morethan 5,000, no more than 2,000 target loci, or no more than 1 ,000 target loci together in the same reaction mixture.

[0090] In some embodiments, the targeted multiplex amplification comprises amplification of 100 to 20,000, 100 to 10,000, 100 to 5,000, 100 to 2,000, or 100 to 1,000 target loci on one or more chromosomes expected to be disomic in the same reaction mixture. In some embodiments, the targeted multiplex amplification comprises amplification of 100 to 20,000, 100 to 10,000, 100 to 5,000, 100 to 2,000, or 100 to 1,000 target loci on Chromosome 1 in the same reaction mixture, wherein the .

[0091] In some embodiments, step (a) further comprises size selection of the cell-free DNA.

[0092] In some embodiments, the primer pairs are each designed to amplify less than about 100 bp of DNA, less than about 80 bp of DNA, or about 65-80 by of DNA.

[0093] In some embodiments, the target loci comprise single nucleotide polymorphism and / or single nucleotide variant loci. In some embodiments, the target loci comprise indel loci. In some embodiments, the target loci comprise copy number variant loci.

[0094] In some embodiments, step (c) comprises performing a barcoding PCR to add a sequencing tag and a sample- specific barcode to obtain a barcoded sequencing library, pooling a plurality of barcoded sequencing libraries and sequencing the pool of barcoded sequencing libraries together in one sequencing lane.

[0095] In some embodiments, both RhD and RhCE sequencing reads are present for all of the exons and / or introns targeted, and the RhD genotype of the fetus is determined to be RhD positive.

[0096] In some embodiments, only RhCE sequencing reads are present for the exons and / or introns targeted, and the RhD genotype of the fetus is determined to have an RhD deletion and be RhD negative.

[0097] In some embodiments, only RhCE sequencing reads are present for exons 4-7 of the RhD gene, whereas both RhD and RhCE sequencing reads are present for the remaining exons targeted, and the RhD genotype of the fetus is determined to be RhD-CE-D hybrid.

[0098] In some embodiments, sequencing reads corresponding to a duplication insert and / or a nucleotide change that creates a stop codon arc present, and the RhD genotype of the fetus is determined to be RhD pseudogene.

[0099] In some embodiments, the pregnant person is at a gestational age of at least 9 weeks or between 9 and 20 weeks.

[0100] Selection of Target Loci

[0101] The RhD and RhCE gene loci are located on human chromosome 1 and are a result of an ancient gene duplication event. As can be seen in FIG. 1A, the RhD gene is flanked upstream and downstream by a Rhesus box. The RhD and RhCE genes have 97% gene identity, differing in only about 36 of their 417 amino acids.

[0102] As shown in FIG. 1, a RhD negative genotype can occur as a result of different mechanisms. As shown in FIG. IB, the entire RhD gene, including portions of the Rhesus boxes, may be deleted, resulting in a hybrid Rhesus box. Alternatively, RhD deletion may occur due to a RhD*psi (or pseudo gene) which has a 37 bp duplication as well as several point mutations relative to RhD, including a nonsense mutation that results in a premature stop codon (FIG. 1C). A RhD negative genotype may also be caused by a RHD-CE-D hybrid, which was generated by an ancestral gene conversion event wherein exons 4-7 of the RhD gene were replaced with exons 4- 7 of RhCE (FIG. ID).

[0103] Due to the similarity of the RhD and RhCE gene sequences, primers were designed to target several introns and exons of the RhD and RhCE gene. For example, in some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten of the primer pairs each targets a different exon or intron of the RhD and RhCE genes and is each designed to amplify a target locus that comprises a variant between the RhD gene and the RhCE homologue gene.

[0104] In some embodiments, at least two, at least three, at least four, or at least five of the primer pairs each targets a different exon of the RhD and RhCE genes and is each designed to amplify a target locus that comprises a variant between the RhD gene and the RhCE homologue gene. In some embodiments, a target locus in exon 1 of the RhD and RhCE genes that comprisesa variant between the RhD gene and the RhCE homologue gene is amplified and sequenced. In some embodiments, a target locus in exon 4 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene is amplified and sequenced. In some embodiments, a target locus in exon 5 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene is amplified and sequenced. In some embodiments, a target locus in exon 7 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene is amplified and sequenced. In some embodiments, a target locus in exon 9 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene is amplified and sequenced.

[0105] In some embodiments, at least one of the primer pair targets a different exon of the RhD and RhCE genes and is designed to amplify a target locus that comprises a nonsense variant between the RhD gene and the RhCE homologue gene. In some embodiments, a target locus in exon 6 of the RhD and RhCE genes that comprises a nonsense variant between the RhD gene and the RhD pseudogene is amplified and sequenced.

[0106] In some embodiments, at least two, at least three, at least four, or at least five of the primer pairs each targets a different intron of the RhD and RhCE genes and is each designed to amplify a target locus that comprises a variant between the RhD gene and the RhCE homologue gene. In some embodiments, a target locus in intron 1 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene is amplified and sequenced. In some embodiments, a target locus in intron 3 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene is amplified and sequenced. In some embodiments, a target locus in intron 4 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene is amplified and sequenced. In some embodiments, a target locus in intron 5 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene is amplified and sequenced. In some embodiments, a target locus in intron 7 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene is amplified and sequenced. In some embodiments, a target locus in intron 8 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene is amplified and sequenced.

[0107] Biological Sample Collection and Preparation

[0108] The cell-free nucleic acid may be isolated from any type of suitable liquid biological specimen or sample (e.g., a test sample). A sample or test sample can be any specimen that is isolated or obtained from a subject or part thereof (e.g., a human subject, a pregnant female, a fetus). In some embodiments, a liquid biological sample is a blood, plasma or serum sample. The term “blood” as used herein refers to a blood sample or preparation from a pregnant person. Blood serum refers to the watery portion of fluid remaining after a blood sample has coagulated. Fluid samples often are collected in accordance with standard protocols hospitals or clinics generally follow. For blood, an appropriate amount of peripheral blood (e.g., between 3-40 milliliters) often is collected and can be stored according to standard procedures prior to or after preparation. A fluid sample from which nucleic acid is extracted may be acellular (e.g., cell-free). In some embodiments, a fluid or tissue sample may contain cellular elements or cellular remnants. In some embodiments, fetal cells or cancer cells may be included in the sample. In some embodiments the cells, cellular elements or cellular remnants are removed from the liquid sample prior to nucleic acid extraction. In a preferred embodiment, 20 ml of blood is collected (10ml in each of 2 tubes) and is stored at room temperature, and the plasma must be isolated within 9 days of blood collection and the cfDNA may be extracted from the plasma.

[0109] For prenatal applications of technology described herein, fluid sample can be collected from a female at a gestational age of at least 9 weeks. Suitable gestational age may vary depending on the prenatal test being performed. In certain embodiments, a pregnant female subject sometimes is in the first trimester of pregnancy, at times in the second trimester of pregnancy, or sometimes in the third trimester of pregnancy.

[0110] Methods for preparing serum or plasma from maternal include, for example, placing a pregnant person's blood in a tube containing EDTA or other substances that allow the isolation of high-quality cell-free DNA, and plasma can then be obtained from whole blood through centrifugation. Serum may be obtained with or without centrifugation-following blood clotting. If centrifugation is used then it is typically, though not exclusively, conducted at an appropriate speed, e.g., 1,500-3,000 times g. Plasma or serum may be subjected to additional centrifugation steps before being transferred to a fresh tube for cfDNA extraction. In some embodiments, cfDNAcan be extracted using a kit. For example, the Qiagen Circulating Nuclei Acid Kit can be used. In some embodiments, the QIAsymphony circulating DNA Kit may be used for automated cfDNA extraction. In some embodiments, gDNA may be extracted using the QIAgen DNeasy Blood and Tissue Kit.

[0111] In some embodiments, the method includes isolating or purifying the DNA. There are a number of standard procedures known in the art to accomplish such an end. In some embodiments, the sample may be centrifuged to separate various layers. In some embodiments, the DNA may be isolated using filtration. In some embodiments, the preparation of the DNA may involve amplification, separation, purification by chromatography, liquid separation, isolation, preferential enrichment, preferential amplification, targeted amplification, or any of a number of other techniques either known in the art or described herein. In some embodiments for the isolation of DNA, RNase is used to degrade RNA. In some embodiments, an QIAamp™ DNA Mini Kit (Qiagen), is used to isolate DNA according to the manufacturer's protocol. In some embodiments, cfDNA molecules are isolated using MagMAXTM cell-free DNA isolation kit (Applied Biosystems). The concentration and purity of DNA may optionally be determined using Nanovue (GE Healthcare, Piscataway, N.J., USA), and DNA integrity may optionally be measured by use of the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif., USA).

[0112] In some embodiments, adaptors are added to make a sequencing library. Prior to ligation, sample DNA may be blunt ended, and then a single adenosine base is added to the 3-prime end. In some embodiments, ligation of adaptors to nucleic acids is a sticky end ligation. Prior to ligation the DNA may be cleaved using a restriction enzyme or some other cleavage method. During ligation the 3-prime adenosine of the sample fragments and the complementary 3-prime tyrosine overhang of adaptor can enhance ligation efficiency. In some embodiments, adaptor ligation is performed using the ligation kit found in the AGILENT SURESELECT™ kit. In some embodiments, the adapters are incorporated into the sequences by PCR. In some embodiments, the adapters are incorporated into the sequences during universal or targeted amplification.

[0113] In some embodiments, the library is amplified using universal primers. In an embodiment, the amplified library is fractionated by size separation or by using products such asAGENCOURT AMPURE™ beads or other similar methods. In some embodiments, PCR amplification is used to amplify target loci. In some embodiments, the amplified DNA is sequenced (such as sequencing using an ILLUMINA IIGAX™ or HiSeq sequencer). In some embodiments, the amplified DNA is sequenced from each end of the amplified DNA to reduce sequencing errors. If there is a sequence error in a particular base when sequencing from one end of the amplified DNA, there is less likely to be a sequence error in the complementary base when sequencing from the other side of the amplified DNA (compared to sequencing multiple times from the same end of the amplified DNA).

[0114] In some embodiments, the sample can include a blood, serum, or plasma sample. In some embodiments, the sample can include free floating DNA in a blood, sera, or plasma sample. In these embodiments, the sample is typically from an animal, such as a mammal or human, and is typically present in fragments about 160 nucleotides in length. In some embodiments, the free- floating DNA is isolated from blood using an EDTA-2Na tube after removal of cellular debris and platelets by centrifugation. The plasma samples can be stored at -80 °C until the DNA is extracted using, for example, QIAamp™ DNA Mini Kit (Qiagen, Hilden, Germany), (e.g. Hamakawa et al., Br J Cancer. 2015; 112:352-356).

[0115] Kits especially adapted for preparing libraries from small nucleic acid fragments, especially circulating cell-free DNA, can be useful for practicing methods provided herein. For example, the NEXTflex™ Cell Free kits (Bioo Scientific, Austin, Tex.) or the Natera Library Prep Kit (Natera, San Carlos, Calif.). Such kits would typically be modified to include adaptors that are customized for the amplification and sequencing steps of the methods provided herein. Adaptor ligation can also be performed using commercially available kits such as the ligation kit found in the Agilent SureSelect™ kit (Agilent, Santa Clara, Calif.).

[0116] Sample nucleic acid molecules are composed of naturally occurring or non-naturally occurring ribonucleotides or deoxyribonucleotides linked through phosphodiester linkages. Furthermore, sample nucleic acid molecules are composed of a nucleic acid segment that is targeted for sequencing. Sample nucleic acid molecules can be or can include nucleic acid segments that are at least 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1,000 nucleotides in length. In any of the embodiments disclosed herein the samplenucleic acid molecules or nucleic acid segments can be between 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, and 500 nucleotides in length on the low end of the range and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, and 10,000 nucleotides in length on the high end of the range. In some embodiments, the nucleic acid molecules can be fragments of genomic DNA and can be between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, and 500 nucleotides in length on the low end of the range and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, and 10,000 nucleotides in length on the high end of the range. For the sake of clarity, nucleic acids initially isolated from a living tissue, fluid, or cultured cells, can be much longer than sample nucleic acid molecules processed using methods herein. As discussed herein, for example, such initially isolated nucleic acid molecules can be fragmented to generate nucleic acid segments, before being used in the methods herein. In some embodiments, the nucleic acid molecules and nucleic acid segments can be identical. The sample nucleic acid molecule or sample nucleic acid segment can include a target locus that contains the nucleotide or nucleotides that are being queried, especially a single nucleotide polymorphism or single nucleotide variant. In any of the disclosed embodiments, the target loci can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1,000 nucleotides in length and include a portion of or the entirety of the sample nucleic acid molecule and / or the sample nucleic acid segment. In other embodiments, the target loci can be between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, and 500 nucleotides in length on the low end of the range and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, and 10,000 nucleotides in length on the high end of the range. In some embodiments, the target loci on different sample nucleic acid molecules can be at least 50%, 60%, 70%, 80%, 90% 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical. In some embodiments, the target loci on different sample nucleic acid molecules can share at least 50%, 60%, 70%, 80%, 90% 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity.

[0117] In some embodiments, the entire sample nucleic acid molecule is a sample nucleic acid segment. For example, in certain embodiments where adaptors arc ligated directly to the ends of sample nucleic acid molecules, or ligated to a nucleic acid(s) ligated to the ends of sample nucleic acid molecules, or ligated as part of primers that bind to sequences at the termini of sample nucleic acid segments, or adapters, such as universal adapters added thereto, as discussed further herein, the entire nucleic acid molecule can be a sample nucleic acid segment. In other embodiments, for example certain embodiments where adaptors are attached to sample nucleic acid molecules as pail of primers that target binding sites internal to the termini of sample nucleic acid molecules, a portion of the sample nucleic acid molecule can be the sample nucleic acid segment that is targeted for downstream sequencing. For example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of a sample nucleic acid molecule can be a nucleic acid segment.

[0118] In some embodiments, sample nucleic acid molecules are a mixture of nucleic acids isolated from a natural source, some sample nucleic acid molecules having identical sequences, some having sequences sharing at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity, and some with less than 50%, 40%, 30%, 20%, 10%, or 5% sequence identity over between 20, 25, 50, 75, 100, 125, 150, 200, 250 nucleotides on the low end of the range, and 50, 75, 100, 125, 150, 200, 250, 300, 400, or 500 nucleotides on the high end of the range. Such sample nucleic acid molecules can be nucleic acid samples isolated from tissues or fluids of a mammal, such as a human, without enriching one sequence over another. In other embodiments, target sequences, for example, those from a gene of interest, can be enriched prior to performing methods provided herein.

[0119] Multiplex Amplification and Sequencing

[0120] In some embodiments, the method comprises performing a multiplex targeted amplification reaction to amplify a plurality of target loci in one reaction mixture before determining the sequences of the amplicons. In certain illustrative embodiments, the nucleic acid sequence data is generated by performing high throughput sequencing of a plurality of copies of a series of amplicons generated using a multiplex amplification reaction, wherein each amplicon of the series of amplicons spans at least one polymorphic locus of the set of polymorphic lociand wherein each of the polymeric loci of the set is amplified. For example, in these embodiments a multiplex PCR to amplify amplicons across at least 10; 100; 200; 500; 1,000; 2,000; 5,000; 10,000; 20,000; 50,000; or 100,000 polymorphic loci (e.g., SNP loci) may be performed. This multiplex reaction can be set up as a single reaction or as pools of different subset multiplex reactions. The multiplex reaction methods provided herein, such as the massive multiplex PCR disclosed herein provide an exemplary process for carrying out the amplification reaction to help attain improved multiplexing and therefore, sensitivity levels.

[0121] In some embodiments, amplification is performed using direct multiplexed PCR, sequential PCR, nested PCR, doubly nested PCR, one-and-a-half sided nested PCR, fully nested PCR, one sided fully nested PCR, one-sided nested PCR, hemi-nested PCR, hemi-nested PCR, triply hemi-nested PCR, semi-nested PCR, one sided semi-nested PCR, reverse semi-nested PCR method, or one-sided PCR, which are described in US Application No. 13 / 683,604, filed Nov.21, 2012, U.S. Publication No. 2013 / 0123120, U.S. Application No. 13 / 300,235, filed Nov. 18, 2011, U.S. Publication No 2012 / 0270212, and U.S. Serial No. 61 / 994,791, filed May 16, 2014, all of which are hereby incorporated by reference in their entirety.

[0122] In some embodiments, multiplex PCR is used. In some embodiments, the method of amplifying target loci in a nucleic acid sample involves (i) contacting the nucleic acid sample with a library of primers that simultaneously hybridize to at least 10; 100; 200; 500; 1 ,000;2,000; 5,000; 10,000; 20,000; 50,000; or 100,000 different target loci to produce a single reaction mixture; and (ii) subjecting the reaction mixture to primer extension reaction conditions (such as PCR conditions) to produce amplified products that include target amplicons. In some embodiments, at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5% of the targeted loci are amplified. In various embodiments, less than 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1, or 0.05% of the amplified products are primer dimers, hi some embodiments, the primers are in solution (such as being dissolved in the liquid phase rather than in a solid phase). In some embodiments, the primers are in solution and are not immobilized on a solid support. In some embodiments, the primers are not part of a microarray.

[0123] In certain embodiments, the multiplex amplification reaction is performed under limiting primer conditions for at least 1 / 2 of the reactions. In some embodiments, limiting primerconcentrations are used in 1 / 10, 1 / 5, 1 / 4, 1 / 3, 1 / 2, or all of the reactions of the multiplex reaction.Provided herein arc factors to consider in achieving limiting primer conditions in an amplification reaction such as PCR.

[0124] In certain embodiments, the multiplex amplification reaction can include, for example, between 2,500 and 50,000 multiplex reactions. In certain embodiments, the following ranges of multiplex reactions are performed: between 10, 100, 200, 250, 500, 1,000, 2,500, 5,000, 10,000, 20,000, 25000, 50,000 on the low end of the range and between 200, 250, 500, 1,000, 2,500, 5,000, 10,000, 20,000, 25,000, 50,000, and 100,000 on the high end of the range.

[0125] In an embodiment, a multiplex PCR assay is designed to amplify potentially heterozygous SNP or other polymorphic or non-polymorphic loci on one or more chromosomes and these assays are used in a single reaction to amplify DNA. The number of PCR assays may be between 10 and 200 PCR assays, between 200 and 1,000 PCR assays, between 1,000 and 5,000 PCR assays, or between 5,000 and 20,000 PCR assays (10 to 200-plex, 200 to 1,000-plex, 1,000 to 5,000-plex, 5,000 to 20,000-plex, more than 20,000-plex respectively). In an embodiment, a multiplex pool of at least 10,000 PCR assays (10,000-plex) are designed to amplify potentially heterozygous SNP loci a single reaction to amplify cfDNA obtained from a blood, plasma, serum, solid tissue, or urine sample. The SNP frequencies of each locus may be determined by clonal or some other method of sequencing of the amplicons. In another embodiment the original cfDNA samples is split into two samples and parallel 5,000-plex assays are performed. In another embodiment the original cfDNA samples is split into n samples and parallel (~10,000 / n)-plex assays are performed where n is between 2 and 12, or between 12 and 24, or between 24 and 48, or between 48 and 96.

[0126] In an embodiment, a method disclosed herein uses highly efficient highly multiplexed targeted PCR to amplify DNA followed by high throughput sequencing to determine the allele frequencies at each target locus. One technique that allows highly multiplexed targeted PCR to perform in a highly efficient manner involves designing primers that are unlikely to hybridize with one another. The PCR probes, typically referred to as primers, are selected by creating a thermodynamic model of potentially adverse interactions between at least 10, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000,or at least 50,000 potential primer pairs, or unintended interactions between primers and sample DNA, and then using the model to eliminate designs that arc incompatible with other the designs in the pool. Another technique that allows highly multiplexed targeted PCR to perform in a highly efficient manner is using a partial or full nesting approach to the targeted PCR. Using one or a combination of these approaches allows multiplexing of at least 10, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, or at least 50,000 primers in a single pool with the resulting amplified DNA comprising a majority of DNA molecules that, when sequenced, will map to targeted loci. Using one or a combination of these approaches allows multiplexing of a large number of primers in a single pool with the resulting amplified DNA comprising greater than 50%, greater than 80%, greater than 90%, greater than 95%, greater than 98%, or greater than 99% DNA molecules that map to targeted loci.

[0127] Bioinformatics Analysis

[0128] In some embodiments, string-to-string alignment of DNA sequences and quality checks can be performed. Aligned sequence data can be processed for determining RhD antigen status. For example, the RHD genotype of each pregnant patient can be identified by calculating an RhD ratio between the amplicons targeting the RHD gene and the RHCE homologue gene (RHCE homologue genes are expected to always be present). In some embodiment, the RhD ratio is calculated as: RhD reads / (RhD reads + RhCE reads). To determine the fetal RhD status, the pregnant patient’s RHD genotype must have been negative. The copy number of the fetus can be determined by using the observed RhD ratio and the expected RhD ratio. If the fetus has at least one detectable copy of RHD (the fetus could have 2 copies if it is an egg donor pregnancy), the algorithm would determine if those copies are a fully intact RHD gene, RHD-CE-D hybrid, or RHD E. The expected RhD ratio takes into consideration the fetal fraction of each sample estimated through a probability model using SNPs that are deemed homozygous in the maternal alleles. Using this genotyping approach allows for the assignment of a confidence based on the probability distribution functions of the different hypotheses and to calculate Chi-squared p-values.

[0129] Statistical Analysis. Summary statistics can be performed on subjects’ demographic and clinical variables. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and no-call rate can be calculated along with corresponding 95% Wilson-Scoreconfidence intervals for the cohort where fetal Rh status is determined. Data analysis can be performed using R version 4.3.3 on RStudio version 2023.12.1+402. Open-source R libraries binom vl.1-1.1, scales vl.3.0, tidyverse v2.0.0, and writexl vl.5.0 corresponding to that R version can be used for data wrangling, data analysis, and report generation.

[0130] In some embodiments, the method described herein has a sensitivity of at least 95%, at least 97%, at least 98%, or at least 99% in determining Rhesus incompatibility. In some embodiments, the method described herein has a specificity of at least 95%, at least 97%, at least 98%, or at least 99% in determining Rhesus incompatibility. In some embodiments, the method described herein has a PPV of at least 95%, at least 97%, at least 98%, or at least 99% in determining Rhesus incompatibility. In some embodiments, the method described herein has a NPV of at least 95%, at least 97%, at least 98%, or at least 99% in determining Rhesus incompatibility.

[0131] In some embodiments, the method described herein accurately identifies at least 95%, at least 97%, at least 98%, or at least 99% of fetal RhD-positive cases. In some embodiments, method described herein accurately identifies at least 95%, at least 97%, at least 98%, or at least 99% of fetal RhD-negative cases. In some embodiments, the method described herein accurately identifies at least 95%, at least 97%, at least 98%, or at least 99% of fetal RhD deletion genotype. In some embodiments, the method described herein accurately identifies at least 95%, at least 97%, at least 98%, or at least 99% of fetal RhD-CE-D hybrid genotype. In some embodiments, the method described herein accurately identifies at least 95%, at least 97%, at least 98%, or at least 99% of fetal RHD* genotype.Methods for Treatment of Rhesus Incompatibility

[0132] The present disclosure also provides method for treatment of Rhesus incompatibility. In one aspect, the present disclosure provides a method of treatment of Rhesus incompatibility, comprising: (a) extracting cell-free DNA from a blood, plasma, or serum sample of a pregnant person who is RhD negative, wherein the extracted DNA comprises a mixture of maternal cell- free DNA and fetal cell-free DNA; (b) performing targeted multiplex amplification on the extracted DNA to amplify a plurality of target loci together in the same reaction mixture using a plurality of primer pairs, wherein at least three of the primer pairs each targets a different exon orintron of the RhD and RhCE genes and is each designed to amplify a target locus that comprises a variant between the RhD gene and the RhCE homologue gene; (c) sequencing the amplicons by high-throughput sequencing to generate sequencing reads of the target loci and determine the RhD genotype of the fetus and the presence or absence of Rhesus incompatibility between the pregnant person and the fetus; and (d) administering Rh immune-glob ulin to the pregnant person determined to have Rhesus incompatibility between the pregnant person and the fetus.

[0133] Current methods of managing RhD incompatibility include providing prophylactic treatment in a RhD negative mother, regardless of the RhD status of the fetus. A Rh immunoglobulin injection is given to the mother at 28 weeks of gestation. Further Rh immune-globulin injections are also required if a miscarriage occurs or any other bleeding events during the pregnancy. After the birth of the child, if the RhD status of the child is determined to be positive, a second shot is administered to the mother. The methods provided herein, will allow for the reduction of Rh immune-globulin injections, only to those RhD negative mothers carrying an RhD positive child.

[0134] In some embodiments, RhD incompatibility is treated with Rh immuno-globulin at around 28 weeks gestation. In some embodiments, Rh incompatibility is treated with Rh immune-globulin within 72 hours of giving birth. In some embodiments, Rh incompatibility is treated with Rh immune-globulin within 72 hours of a possible exposure of the mother to Rh positive blood, for example after miscarriage of a Rh positive fetus by a Rh negative mother.

[0135] In some embodiments, mothers of pregnancies determined to be Rh incompatible can also be tested for the presence of Rh antibodies. The presence of Rh antibodies in a Rh negative mother indicates that the mother has been exposed to Rh positive red blood cells from either this, or a previous Rh positive pregnancy. The presence of Rh antibodies while carrying a Rh negative fetus could lead to severe hemolytic disease in the fetus and newborn. Therefore testing pregnancies determined to be Rh incompatible for the presence of Rhesus antibodies will allow for appropriate treatment plans to be put in place, and therefore reduce the likelihood of miscarriage in a Rh incompatible pregnancy.Kits

[0136] The present disclosure also provides kits for determining Rh incompatibility. In some embodiments, the kit comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten primer pairs each targeting a different exon or intron of the RhD and RhCE genes and each designed to amplify a target locus that comprises a variant between the RhD gene and the RhCE homologue gene.

[0137] In some embodiments, the kit comprises at least two, at least three, at least four, or at least five primer pairs each targeting a different exon of the RhD and RhCE genes and each designed to amplify a target locus that comprises a variant between the RhD gene and the RhCE homologue gene. In some embodiments, the kit comprises a primer pair designed to amplify a target locus in exon 1 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene. In some embodiments, the kit comprises a primer pair designed to amplify a target locus in exon 4 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene. In some embodiments, the kit comprises a primer pair designed to amplify a target locus in exon 5 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene. In some embodiments, the kit comprises a primer pair designed to amplify a target locus in exon 7 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene. In some embodiments, the kit comprises a primer pair designed to amplify a target locus in exon 9 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene.

[0138] In some embodiments, the kit comprises at least one primer pair targeting a different exon of the RhD and RhCE genes and designed to amplify a target locus that comprises a nonsense variant between the RhD gene and the RhCE homologue gene. In some embodiments, the kit comprises a primer pair designed to amplify a target locus in exon 6 of the RhD and RhCE genes that comprises a nonsense variant between the RhD gene and the RhD pseudogene.

[0139] In some embodiments, the kit comprises at least two, at least three, at least four, or at least five primer pairs each targeting a different intron of the RhD and RhCE genes and each designed to amplify a target locus that comprises a variant between the RhD gene and the RhCE homologue gene. In some embodiments, the kit comprises a primer pair designed to amplify a target locus inintron 1 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene. In some embodiments, the kit comprises a primer pair designed to amplify a target locus in intron 3 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene. In some embodiments, the kit comprises a primer pair designed to amplify a target locus in intron 4 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene. In some embodiments, the kit comprises a primer pair designed to amplify a target locus in intron 5 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene. In some embodiments, the kit comprises a primer pair designed to amplify a target locus in intron 7 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene. In some embodiments, the kit comprises a primer pair designed to amplify a target locus in intron 8 of the RhD and RhCE genes that comprises a variant between the RhD gene and the RhCE homologue gene.

[0140] The kit may further comprise one or more of: wash buffers and / or reagents, hybridization buffers and / or reagents, labeling buffers and / or reagents, dilution buffers and / or reagents, and detection means. The buffers and / or reagents are usually optimized for the particular amplification / detection technique for which the kit is intended. Protocols for using these buffers and reagents for performing different steps of the procedure may also be included in the kit.

[0141] The kit additionally may comprise an assay definition scan card and / or instructions such as printed or electronic instructions for using the oligonucleotides in an assay. In some embodiments, a kit comprises an amplification reaction mixture or an amplification master mix. Reagents included in the kit may be contained in one or more containers, such as a vial.WORKING EXAMPLESExample 1 - Clinical Validation of a Prenatal Cell-free DNA Screening Test for Fetal RHD in a Large US Cohort

[0142] Introduction: Rh immune globulin prophylaxis is standard of care for Rh-negative pregnant patients in the US. Internationally, prenatal cell- free DNA screening (cfDNA) for fetal Rh status provides benefits for Rh-negative pregnant patients by reducing invasive diagnostic testing andintensity of surveillance for alloimmunized individuals and by reducing blood product use in non- alloimmunizcd pregnancies.

[0143] Methods: This clinical validation study assessed the performance of a commercially available, prenatal next-generation sequencing (NGS)-based cfDNA test for fetal RHD status in a large US cohort of Rh-negative pregnant patients. Samples were identified from individuals who previously had SNP-based cfDNA for fetal aneuploidy at a commercial laboratory. Maternal and fetal RHD genotypes were evaluated through prospective cfDNA NGS analysis. Serological diagnostic confirmation of maternal and newborn Rh blood type was available for all patients. Investigators were blinded to fetal Rh status.

[0144] Results: The cohort consisted of 655 pregnant patients with serological results for Rh antigen. Patient demographics included a representative mix of race and ethnicities in the Rh- negative US population (2.1% Asian, 7.0% Black, 74.0% White, and 13.7% Hispanic). cfDNA fetal RHD was reported in 653 / 655; 2 cases were not called. There were 0 false-negative cases identified with 356 / 356 fetuses correctly identified as fetal Rh-positive (sensitivity: 100% [95%CI: 98.9- 100.0]). Of the 297 Rh-negative fetuses, 295 were correctly identified as Rh-negative and 2 were false-positives (specificity: 99.3% [95%CI: 97.6-99.8]). Of the fetuses with a negative Rh phenotype, the cfDNA test accurately identified 3 with RHD genotype.

[0145] Conclusions: Validation of this test in the largest US cohort of Rh-ncgativc patients provides data on early and accurate noninvasive prenatal identification of fetal RHD genotype to potentially assist patients and providers in prevention and management of Rh alloimmunization.

[0146] Alloimmunization, can occur during pregnancy from mixing of maternal with fetal red blood cells. Rh alloimmunization refers to the maternal formation of antibodies against fetal Rh and can occur in Rh-negative pregnant patients carrying Rh-positive fetus(es). Administration of Rh immune globulin (RhIG) to all Rh-negative patients is the current standard of care in the US to reduce the risk of alloimmunization. Given the recent RhIG supply constraints in the US, noninvasive prenatal testing for fetal Rh incompatibility has the potential for increased utility in the US. As such, ACOG and SMFM have highlighted cell-free DNA (cfDNA) screening for fetal RHD genotype as a reasonable option and in particular for alloimmunized pregnancies.

[0147] Early detection of fetal Rh status can help guide RhIG use in Rh-negative pregnancies and reduce the need for invasive procedures and periodic surveillance for alloimmunizcd pregnant individuals. We present the largest US clinical validation of a next-generation sequencing (NGS)- based, non-invasive prenatal cfDNA test for fetal RHD.

[0148] Assay Design. A NGS-based non-invasive prenatal cfDNA test for fetal RHD genotype was developed for use in pregnant patients with a Rh-negative phenotype based on serological testing as part of routine antenatal care. In general, an Rh-positive phenotype based on serological testing is associated with expression of Rh antigens on the surface of red blood cells while an Rh- negative phenotype is associated with absence of expression of the Rh antigen on red blood cells and the potential for Rh alloimmunization on exposure to Rh antigens.

[0149] The Rh phenotype is the result of changes in the RHD gene located on Chromosome 1. The RHD gene likely arose as the result of an ancient duplication of the adjacent RHCE gene. As a result, the two genes are very similar with 92% nucleotide sequence homology between the RHD and RHCE genes across all 10 exons and introns. A Rh-negative phenotype has been reported as a result of hundreds of different variants in the RHD gene but the three most commonly reported genotypes are: 1) RHD deletion, where the entire RHD gene is deleted, and a hybrid Rh box containing portions of two terminal Rh boxes is present; 2) RHD-CE-D hybrid, where RHD gene fragments are replaced with their RHCE counterparts; and 3) RHD pseudogene (RHD'P), an inactive RHD gene with a insertion, frameshift mutation, three missense mutations, and a nonsense mutation, which ultimately prevents translation of the gene.

[0150] A multiplex-PCR NGS-based screening assay was developed by designing primers to regions of the RHD gene that differed from the RHCE homologue gene and distinguished the RHD'P. The amplicons were targeted to regions of RHD that allow identification of the following fetal genotypes and subsequent predicted phenotype (in parentheses): RHD (positive); RHD deletion (negative); RHD'P (negative); and RHD-CE-D hybrid (negative). The fetal RHD test is designed to detect all known weak D and Partial D genotypes including a majority of hybrid partial D genotypes to avoid the possibility of obtaining false negative results, which, while rare, can occur with most routine qPCR screening assays.

[0151] Selection of Cases with Serology Truth. Clinical cases with previous commercial SNP- bascd non-invasivc prenatal cfDNA testing that was performed between April 1, 2022 to September 30, 2022 with available residual samples were identified and matched to clinical outcomes collected for quality assurance where serological Rh phenotypes were available for both the pregnant patient and their baby. Inclusion criteria for use in this clinical validation were: a previously reported non-invasive prenatal cfDNA result for fetal aneuploidy; the residual sample passed quality metrics; documented Rh-negative serological results for the pregnant person; documented serological results for the Rh antigen in the fetus or newborn for the pregnancy of interest (serological truth); maternal genotype was identified as RHD deletion or RHD-CE-D hybrid; and the pregnancy was identified as a singleton or monozygotic twin pregnancy based on SNP-based non-invasive prenatal cfDNA testing. Cases were excluded from the clinical validation of fetal RHD if the pregnant patient ‘opted-out’ of future use of their clinical sample for research. Demographics and clinical characteristics were recorded. All linked data and samples were deidentified prior to any research activities. This study was reviewed by Salus IRB and designated an exempt protocol (Protocol 17-035 Prenatal) with a waiver of patient consent.

[0152] Sample Preparation and Sequencing. Samples were initially processed for commercial non-invasive prenatal cfDNA testing as previously described. Specifically, a set of primers (13 pairs) for targeted amplicons covering regions differing between the RHD and RHCE genes were designed and incorporated into the SNP-based NIPT assay - Panorama. RHD and RHCE can be directly detected by targeting the differences between these two highly similar genes. This assay can accurately detect fetal RHD status, including different RHD- genotypes (RHDdel, RHD- RHCE hybrid and RHDpsi etc.), at fetal fraction >= 2.8%. Notably, Panorama as say / algorithm can precisely detect fetal fraction. Residual sequencing libraries made from cfDNA for each of the included cases were sequenced on a NOVAseq 6000 (Illumina, San Diego, CA) using S4 flowcell with an average read depth of >8 million.

[0153] Assay Bioinformatics. String-to-string alignment of DNA sequences and quality checks were performed as previously described. Aligned sequence data were then processed for determining Rh antigen status. In brief, the algorithm identified the RHD genotype of each pregnant patient by calculating an RhD ratio between the amplicons targeting the RHD gene and the RHCE homologue gene (RHCE homologue genes are expected to always be present). In someembodiment, the RhD ratio is calculated as: RhD reads / (RhD reads + RhCE reads). To determine the fetal Rh status, the pregnant patient’s RHD genotype must have been negative. The algorithm then determined the copy number of the fetus by using the observed RhD ratio and the expected RhD ratio. If the fetus had at least one detectable copy of RHD (the fetus could have 2 copies if it were an egg donor pregnancy) the algorithm would determine if those copies were a fully intact RHD gene, RHD-CE-D hybrid, or RHD'IJ. The expected RhD ratio takes into consideration the fetal fraction of each sample estimated through a probability model using SNPs that were deemed homozygous in the maternal alleles. Using this genotyping approach allowed for the assignment of a confidence based on the probability distribution functions of the different hypotheses and to calculate Chi-squared p-values. The algorithm was blinded to sample status, and all calls were reported as predicted by the algorithm, without subjective modification by laboratory personnel.

[0154] Statistical Analysis. Summary statistics were performed on subjects’ demographic and clinical variables. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and no-call rate were calculated along with corresponding 95% Wilson-Score confidence intervals for the cohort where fetal Rh status was determined. Data analysis was performed using R version 4.3.3 on RStudio version 2023.12.1+402. Open-source R libraries binom vl.1-1.1, scales vl.3.0, tidyverse v2.0.0, and writexl vl.5.0 corresponding to that R version were used for data wrangling, data analysis, and report generation.

[0155] Results. The final cohort that met eligibility criteria for screening for fetal RHD consisted of samples from 655 pregnant patients. Median maternal age was 32 (IQR: 27.0 - 35.0) years and median weight was 70 (IQR: 61.0 - 85.0) kg at the time of non-invasive prenatal cfDNA testing (Table 1). Race and ethnicity as documented medical record were 14 (2.1%) Asian, 46 (7.0%) Black, 485 (74.0%) White, 4 (0.6%) other, and 106 (16.2%) unknown race; 90 (13.7%) Hispanic or Latino, 444 (67.8%) Non-Hispanic, and 121 (18.5%) unknown ethnicity. The median gestational age at the time of testing was 12 (IQR: 10.8 - 13.1) weeks, and the median fetal fraction was 8.3% (IQR: 6.0% - 11.0%). Fetal sex was split between males (51.2%) and females (47.8%). Most pregnancies were singleton (99.1%); 5 (0.8%) were monozygotic twin pregnancies, and 1 (0.2%) fetus was conceived through egg donation (Table 1).

[0156] Table 1. Demographics and Pregnancy Characteristics of Study Cohort

[0157] Of 655 samples that had a prenatal cfDNA test for fetal RHD genotype, 2 did not receive a result (1 had a fetal fraction exceeding the 30% upper limit of detection range and 1 had a low confidence call) resulting in a no-call rate estimate of 0.3%. There were 0 false-negative cases identified and 356 / 356 cases were correctly identified as fetal Rh-positive based on serologicaltesting (Table 2), resulting in an assay sensitivity of 100% (95%CI: 98.9 - 100.0, Table 3). Of the 297 Rh-ncgativc fetuses, 295 were correctly identified with prenatal cfDNA testing as fetal RHD negative. Two were false-positive cases (Table 2). Thus, the cfDNA RHD test had a specificity of 99.3% (95%CI: 97.6 - 99.8). Of the fetuses with a negative Rh phenotype, the cfDNA test identified 3 with RHD*'P genotype. The positive predictive value for the test was 99.4% and the negative predictive value was >99.9% (Table 3).

[0158] Table 2. Prenatal cfDNA RHD Assay Results Compared to Serological TruthNIPT = non-invasive prenatal cfDNA testing

[0159] Table 3. Prenatal cfDNA RHD Assay PerformancePPV = positive predictive value; NPV = negative predictive value; Cl = confidence interval; n = cfDNA analysis; N = serological truth

[0160] Discussion. Discerning fetal Rh status is vital for reducing possible risks of alloimmunization and adverse pregnancy outcomes as well as to direct management for Rh- negative pregnancies. Shortage in supply of RhIG, the only immunoprophylactic agent, in the US has prompted the clinical research field to adopt non-invasive fetal RHD testing that is equitable across the diverse racial and ethnic population. Herein we performed the largest clinical validation study, in a demographic ally diverse US population, of a commercially available non-invasive prenatal cfDNA test for fetal RHD. The assay performance was excellent with a sensitivity of 100%, specificity of 99.3%, PPV of 98.4%, and NPV of 100%. Implementation of this test has the potential to avoid unnecessary use of a blood product (RhIG) in up to 40% of Rh-negative pregnant patients and conserve the currently limited US RhIG supply.

[0161] In summary, this study demonstrates that a commercially available non-invasive prenatal cfDNA screening test has high sensitivity and specificity in a diverse US population and has the potential to transform the approach to management of Rh-negative pregnant patients in the US.

Claims

CLAIMSWhat is claimed is:

1. A method of preparing a non-naturally occurring composition comprising:(a) extracting cell-free DNA from a blood, plasma, or serum sample of a pregnant person, wherein the extracted DNA comprises a mixture of maternal cell-free DNA and fetal cell-free DNA;(b) performing targeted multiplex amplification on the extracted DNA to amplify a plurality of target loci together in the same reaction mixture using a plurality of primer pairs, wherein at least three of the primer pairs each targets a different exon or intron of the RhD gene and the RhCE homologue gene and is each designed to amplify a target locus that comprises a variant between the RhD gene and the RhCE homologue gene, said targeted multiplex amplification resulting in a non-naturally occurring composition;(c) sequencing at least some of the amplicons in said non-naturally occurring composition by high-throughput sequencing to generate sequencing reads and determine the RhD genotype of the fetus using the sequencing reads of the amplicons targeting the RhD gene and the RhCE homologue gene.

2. The method of claim 1, wherein the pregnant person is RhD negative.

3. The method of any of the preceding claims, wherein at least five of the primer pairs each targets a different exon or intron of the RhD gene and the RhCE homologue gene and is each designed to amplify a target locus that comprises a variant between the RhD gene and the RhCE homologue gene.

4. The method of any of the preceding claims, wherein at least ten of the primer pairs each targets a different exon or intron of the RhD gene and the RhCE homologue gene and is each designed to amplify a target locus that comprises a variant between the RhD gene and the RhCE homologue gene.

5. The method of any of the preceding claims, wherein a target locus in exon 7 of the RhD gene that comprises a variant between the RhD gene and the RhCE homologue gene is amplified and sequenced.

6. The method of any of the preceding claims, wherein a target locus in exon 9 of the RhD gene that comprises a variant between the RhD gene and the RhCE homologue gene is amplified and sequenced.

7. The method of any of the preceding claims, wherein a target locus in exon 6 of the RhD gene that comprises a nonsense variant between the RhD gene and the RhD pseudogene is amplified and sequenced.

8. The method of any of the preceding claims, wherein the targeted multiplex amplification comprises amplification of at least 100, at least 500, at least 1,000, or at least 10,000 target loci together in the same reaction mixture.

9. The method of any of the preceding claims, wherein the targeted multiplex amplification comprises amplification of 100 to 20,000 target loci on one or more chromosomes expected to be disomic.

10. The method of any of the preceding claims, wherein step (a) further comprises size selection of the cell-free DNA.11 . The method of any of the preceding claims, wherein the primer pairs are each designed to amplify less than about 100 bp of DNA, or less than about 80 bp of DNA, or about 65-80 by of DNA.

12. The method of any of the preceding claims, wherein the target loci comprise single nucleotide polymorphism and / or single nucleotide variant loci.

13. The method of any of the preceding claims, wherein step (c) comprises performing a barcoding PCR to add a sequencing tag and a sample-specific barcode to obtain a barcoded sequencing library, pooling a plurality of barcoded sequencing libraries and sequencing the pool of barcoded sequencing libraries together in one sequencing lane.

14. The method of any of claims 1-13, wherein both RhD and RhCE sequencing reads are present, and the RhD genotype of the fetus is RhD+.

15. The method of any of claims 1-13, wherein only RhCE sequencing reads are present, and the RhD genotype of the fetus is RhD deletion.

16. The method of any of claims 1-13, wherein only RhCE sequencing reads are present for exons 4-7 of the RhD gene, and the RhD genotype of the fetus is RhD-CE-D hybrid.

17. The method of any of claims 1-13, wherein sequencing reads corresponding to a duplication insert and / or a nucleotide change that creates a stop codon are present in the fetus, and the RhD genotype of the fetus is RhD pseudogene.

18. The method of any of the preceding claims, wherein the pregnant person is at a gestational age of between 9 and 20 weeks.

19. A method of preparing a non-naturally occurring composition comprising:(a) genotyping a pregnant person to determine the RhD genotype of the pregnant person;(b) extracting cell-free DNA from a blood, plasma, or serum sample of the pregnant person who is RhD negative, wherein the extracted DNA comprises a mixture of maternal cell- free DNA and fetal cell-free DNA;(c) performing targeted multiplex amplification on the extracted DNA to amplify a plurality of target loci together in the same reaction mixture using a plurality of primer pairs, wherein at least three of the primer pairs each targets a different exon or intron of the RhD gene and the RhCE homologue gene and is each designed to amplify a target locus that comprises a variant between the RhD gene and the RhCE homologue gene, said targeted multiplex amplification resulting in a non-naturally occurring composition;(d) sequencing at least some of the amplicons in said non-naturally occurring composition by high-throughput sequencing to generate sequencing reads and determine the RhD genotype of the fetus using the sequencing reads of the amplicons targeting the RhD gene and the RhCE homologue gene, thereby identifying the presence or absence of Rhesus incompatibility between the pregnant person and the fetus.

20. A method of preparing a non-naturally occurring composition pursuant to treating Rhesus incompatibility, comprising:(a) extracting cell-free DNA from a blood, plasma, or serum sample of a pregnant person who is RhD negative, wherein the extracted DNA comprises a mixture of maternal cell-free DNA and fetal cell-free DNA;(b) performing targeted multiplex amplification on the extracted DNA to amplify a plurality of target loci together in the same reaction mixture using a plurality of primer pairs, wherein at least three of the primer pairs each targets a different exon or intron of the RhD gene and the RhCE homologue gene and is each designed to amplify a target locus that comprises a variant between the RhD gene and the RhCE homologue gene, said targeted multiplex amplification resulting in a non-naturally occurring composition;(c) sequencing at least some of the amplicons in said non-naturally occurring composition by high-throughput sequencing to generate sequencing reads and determine the RhD genotype of the fetus using the sequencing reads of the amplicons targeting the RhD gene and the RhCE homologue gene, thereby identifying the presence or absence of Rhesus incompatibility between the pregnant person and the fetus; and(d) administering Rh immune-globulin to the pregnant person determined to have Rhesus incompatibility between the pregnant person and the fetus.

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