Compositions and methods for identifying chromosomal microdeletions

Abstract

Provided herein are compositions for use as positive controls for detecting one or more targeted microdeletions in a sample. The positive controls can be used to determine the error rate and efficiency of assays used to identify microdeletions, such as 22q11.2 deletions (DiGeorge syndrome), chromosome 5p15.2 (Cri-du-Chat syndrome), 1p36 deletions, 15q11.2-q13 deletions (Prader-Willi syndrome), and / or 15q11-q13 (Angelman syndrome).

Description

[Background technology]

[0001] Subchromosomal abnormalities, such as microdeletions and duplications, can cause severe physical and / or intellectual disabilities. Eight microdeletion syndromes have a combined incidence of more than 1 in 1000, making them almost as common as fetal autosomal trisomies.

[0002] In young women, the risk of clinically significant microdeletions outweighs the risk of Down syndrome. Prenatal testing is important for optimal management, as some infants with subregional chromosomal abnormalities may benefit from early intervention.

[0003] Chromosomal subregion abnormalities, such as microdeletions and duplications, are relatively more difficult to detect than chromosomal abnormalities due to their small size. Improved methods and compositions for detecting chromosomal subregion abnormalities, such as microdeletions, are needed. Summary of the Invention

[0004] The present disclosure provides a positive control composition for detecting microdeletions.

[0005] The present disclosure provides a composition comprising an engineered nucleic acid construct for use as a positive control for detecting one or more microdeletions of interest in a sample, the construct being engineered from a reference nucleic acid sequence comprising the microdeletion of interest, the construct comprising a 5' terminal region, a central region, and a 3' terminal region, the 5' terminal region and the 3' terminal region comprising the reference sequence on either side of the microdeletion of interest, the central region of the construct being a DNA barcode, and the barcode replacing the microdeletion sequence of the reference nucleic acid sequence.

[0006] In some embodiments, the microdeletion of interest corresponds to a 22q11.2 deletion, a 5p15.2 deletion, a 1p36 deletion, a 15q11.2-q13 deletion, or a 15q11-q13 deletion. In some embodiments, the microdeletion of interest is associated with cancer.

[0007] In some embodiments, the composition comprises: (i) a first engineered nucleic acid construct comprising a barcode that replaces a targeted microdeletion corresponding to a 22q11.2 deletion; (ii) a second engineered nucleic acid construct comprising a barcode that replaces a targeted microdeletion corresponding to a 5p15.2 deletion; (iii) a third engineered nucleic acid construct comprising a barcode that replaces a targeted microdeletion corresponding to a 1p36 deletion; (iv) a fourth engineered nucleic acid construct comprising a barcode that replaces a targeted microdeletion corresponding to a 15q11.2-q13 deletion; and (v) a fifth engineered nucleic acid construct comprising a barcode that replaces a targeted microdeletion corresponding to a 15q11-q13 deletion.

[0008] In some embodiments, the barcode is about 6 base pairs to about 9 base pairs.

[0009] In some embodiments, the 5' and 3' terminal regions of the construct contain at least one single nucleotide polymorphism (SNP) of interest.

[0010] In some embodiments, the 5' and 3' terminal regions of the construct contain sequences recognized by primers targeting SNPs within or on either side of the microdeletion of interest.

[0011] In some embodiments, the reference nucleic acid sequence is maternal DNA, and the SNP of interest is altered so that the construct can act as a positive control for the offspring DNA.

[0012] In some embodiments, the size of the construct is from about 100 bp to about 200 bp, or from about 160 bp to about 200 bp.

[0013] In another aspect, the disclosure provides a method for preparing a construct for use as a positive control for detecting a microdeletion of interest in a sample, the method comprising obtaining a reference nucleic acid, isolating a nucleic acid sequence comprising the 5' and 3' ends on either side of the microdeletion of interest, and replacing a central region of the reference nucleic acid sequence corresponding to the microdeletion of interest with a barcode.

[0014] In some embodiments, the reference nucleic acid is obtained from a cell line suitable for use as a positive control for detecting one or more microdeletions.

[0015] In some embodiments, the reference nucleic acid is a mononucleosome. In some embodiments, the reference nucleic acid is genomic DNA.

[0016] In another aspect, the disclosure provides a method for preparing a preparation of amplified DNA from a sample or a portion thereof useful for identifying one or more microdeletions associated with a disease or disorder, comprising: (a) preparing a construct for use as a positive control for detecting one or more microdeletions; (b) adding the construct from (a) to the sample or portion thereof to obtain a spiked sample; and extracting nucleic acid from the spiked sample or portion thereof; (c) performing targeted amplification on the spiked sample or portion thereof from (b) to amplify one or more target regions that include the microdeletions of interest to obtain amplicons; and (d) analyzing the amplicons or portions thereof from (c) to determine (i) whether the amplicons include the construct amplified as a positive control; and (ii) whether the amplicons include the one or more microdeletions of interest.

[0017] In some embodiments, preparation of a construct for use as a positive control for the detection of one or more microdeletions is performed by chemical synthesis followed by PCR amplification of the synthesized construct.

[0018] In some embodiments, the sample is a plasma sample and comprises cell-free DNA. In some embodiments, the plasma sample comprises maternal and fetal cell-free DNA, and the SNPs in the construct are altered to act as a positive control for fetal cell-free DNA. In some embodiments, the sample comprises circulating tumor DNA (ctDNA).

[0019] In some embodiments, at least five microdeletions of interest are amplified in a single reaction volume, and a construct for use as a positive control is prepared for each of the at least five microdeletions of interest. In some embodiments, methods for preparing a preparation of amplified DNA from a sample or a portion thereof useful for identifying one or more microdeletions associated with a disease or disorder disclosed herein include a 22q11.2 deletion (DiGeorge syndrome), a chromosome 5p15.2 (Cri-du-Chat syndrome), a 1p36 deletion, a 15q11.2-q13 deletion (Prader-Willi syndrome), and / or a 15q11-q13 (Angelman syndrome).

[0020] In some embodiments, the methods disclosed herein for preparing a preparation of amplified DNA from a sample or a portion thereof useful for identifying one or more microdeletions associated with a disease or disorder further comprise sequencing to detect (i) the presence of a construct as a positive control, and (ii) the presence of one or more microdeletions of interest.

[0021] In some embodiments, the methods disclosed herein for preparing a preparation of amplified DNA from a sample or portion thereof useful for identifying one or more microdeletions associated with a disease or disorder use positive controls to determine the efficiency and error rate of each amplification reaction, and use the efficiency and error rate to determine the presence of one or more microdeletions of interest.

[0022] In some embodiments, the present disclosure provides that the amount of construct added to a sample is determined by (a) mixing DNA from a normal female cell line and the construct in a range of ratios to generate a titration series to determine the limit of detection, (b) adding the mixture of (a) to DNA-depleted plasma, (c) performing targeted amplification of a microdeletion in which the construct is a positive control, and (d) determining the ratio of the construct to mononucleosomal DNA from the normal cell line that allows detection of the construct. In some embodiments, the DNA is mononucleosomal or genomic DNA.

[0023] In another aspect, the disclosure provides a method of preparing a sample comprising nucleic acid, comprising spiking the sample with a composition. In some embodiments, the sample is a maternal plasma sample.

[0024] In some embodiments, detection of SNPs on either side of the barcode replacing the microdeletion in the engineered construct within the amplicon demonstrates that the assay works to detect SNPs on either side of and within the microdeletion, thereby confirming that the engineered construct can be used as a positive control. [Brief explanation of the drawings]

[0025] [Figure 1] 1 is a graphical representation of an engineered nucleic acid construct for use as a positive control for the detection of microdeletions. MD stands for microdeletion. STAR primers refer to the STAR PCR protocol described elsewhere herein. STAR stands for specific target amplification reaction. [Figure 2] 1 is a flow chart illustrating the use of the engineered nucleic acid constructs disclosed herein as a positive control for the detection of microdeletions. DETAILED DESCRIPTION OF THE INVENTION

[0026] Reference will now be made in detail to some specific embodiments of the invention contemplated by the inventors for carrying out the invention. Certain examples of these specific embodiments are illustrated in the accompanying drawings. While the invention will be described in conjunction with these specific embodiments, it will be understood that it is not intended to limit the invention to the described embodiments. On the contrary, it is intended to cover all alternatives, modifications, and equivalents that may be included.

[0027] The present disclosure provides improved methods for determining targeted microdeletions or other chromosomal subregion abnormalities. In some embodiments, the present disclosure provides methods for non-invasive prenatal testing (NIPT), specifically, methods for identifying microdeletions or other chromosomal subregion abnormalities in a fetus by performing targeted amplification and using an engineered positive control construct disclosed herein that corresponds to the targeted microdeletion.

[0028] In particular, the present disclosure provides a composition comprising an engineered nucleic acid construct for use as a positive control for detecting one or more microdeletions of interest in a sample, the construct being engineered from a reference nucleic acid sequence comprising the microdeletion of interest, the construct comprising a 5' terminal region, a central region, and a 3' terminal region, the 5' terminal region and the 3' terminal region comprising the reference sequence on either side of the microdeletion of interest, and the central region of the construct being a DNA barcode, the barcode replacing the microdeletion sequence of the reference nucleic acid sequence.

[0029] Because the barcode is unique to a particular microdeletion region, a positive control PCR for a particular microdeletion PCR assay can be determined and analyzed. The positive control PCR can be used to determine the efficiency and error rate of the PCR assay for the microdeletion of interest, thereby improving the accuracy of microdeletion testing.

[0030] An illustration of an exemplary engineered nucleic acid construct for use as a positive control for detection of one or more targeted microdeletions as disclosed herein is provided in Figure 1. As shown in Figure 1, the engineered nucleic acid construct comprises a barcode that replaces the region corresponding to the targeted microdeletion and retains the 5' and 3' terminal regions on either side of the region corresponding to the targeted microdeletion. In some embodiments, the DNA barcode can be about 4 to about 10 base pairs, about 5 to about 10 base pairs, 6 to about 10 base pairs, 6 to about 9 base pairs, 6 to about 9 base pairs, about 6 to about 12 base pairs, about 6 to about 15 base pairs, about 6 to about 18 base pairs, about 6 to about 20 base pairs, about 6 base pairs, about 7 base pairs, about 8 base pairs, or about 10 base pairs.

[0031] In some embodiments, the size of the construct is about 100 bp to about 200 bp, about 160 bp to about 200 bp, about 100 bp to about 300 bp, about 100 bp to about 400 bp, or about 100 bp to about 500 bp.

[0032] In some embodiments, the microdeletion of interest corresponds to a 22q11.2 deletion, a 5p15.2 deletion, a 1p36 deletion, a 15q11.2-q13 deletion, or a 15q11-q13 deletion. The 22q11.2 deletion is associated with DiGeorge syndrome. In some embodiments, the microdeletion is associated with Prader-Willi syndrome. In some embodiments, the microdeletion is associated with Angelman syndrome. In some embodiments, the microdeletion is a 1p36 deletion. In some embodiments, the microdeletion is associated with Cri-du-Chat syndrome.

[0033] In some embodiments, the composition comprises: (i) a first engineered nucleic acid construct comprising a barcode replacing a targeted microdeletion corresponding to a 22q11.2 deletion, (ii) a second engineered nucleic acid construct comprising a barcode replacing a targeted microdeletion corresponding to a 5p15.2 deletion, (iii) a third engineered nucleic acid construct comprising a barcode replacing a targeted microdeletion corresponding to a 1p36 deletion, (iv) a fourth engineered nucleic acid construct comprising a barcode replacing a targeted microdeletion corresponding to a 15q11.2-q13 deletion, and (v) a fifth engineered nucleic acid construct comprising a barcode replacing a targeted microdeletion corresponding to a 15q11-q13 deletion. In some embodiments, the composition comprises a plurality of engineered nucleic acid constructs, each construct being a positive control for the targeted microdeletion region. The composition may comprise, at the lower limit, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, or 50 different engineered nucleic acid constructs, and at the upper limit, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 50, 100, or 250 different engineered nucleic acid constructs, each construct being a positive control for the microdeletion region of interest.

[0034] In some embodiments, the 5' and 3' terminal regions of the construct comprise at least one single nucleotide polymorphism (SNP) of interest. In some embodiments, the 5' and 3' terminal regions of the construct comprise sequences recognized by primers targeting SNPs within or on either side of the microdeletion of interest. In some embodiments, the reference nucleic acid sequence is maternal DNA, and the SNP of interest is altered (or inverted) to allow the construct to act as a positive control for the offspring DNA.

[0035] The term "single nucleotide polymorphism (SNP)" refers to a single nucleotide that may differ between the genomes of two members of the same species. The use of this term should not imply any restriction on the frequency with which each variant occurs.

[0036] The term "sequence" refers to a DNA sequence or gene sequence. It can refer to the primary physical structure of a DNA molecule or strand in an individual. It can refer to the sequence of nucleotides found within that DNA molecule, or the complementary strand to a DNA molecule. It can refer to the information contained in a DNA molecule as its representation in a computer.

[0037] The term "locus" refers to a particular region of interest on an individual's DNA, which may refer to a SNP, a site of a possible insertion or deletion, a chromosome or portion thereof, or the site of some other associated genetic variation. A disease-associated SNP may also be referred to as a disease-associated locus.

[0038] The term "polymorphic allele" or "polymorphic locus" refers to an allele or locus whose genotype varies among individuals within a given species. Some examples of polymorphic alleles include single nucleotide polymorphisms, short tandem repeats, deletions, duplications, and inversions.

[0039] In another aspect, the present disclosure provides a method for preparing a construct for use as a positive control for detecting a microdeletion of interest in a sample according to claims 1-8, the method comprising obtaining a reference nucleic acid, isolating a nucleic acid sequence comprising the 5' and 3' ends on either side of the microdeletion of interest, and replacing a central region of the reference nucleic acid sequence corresponding to the microdeletion of interest with a barcode.

[0040] As used herein, the term "nucleic acid" refers to nucleic acid in the broadest sense and is not limited to a particular form or type of nucleic acid. In some embodiments, nucleic acid can be genomic DNA, mononucleosomes, dinucleosomes, trinucleosomes, cell-free DNA, or DNA obtained from a cell, tissue, or organ. Nucleic acid can also refer to any type of RNA, including, but not limited to, small non-coding RNA such as miRNA, tRNA, or piwiRNA. The term nucleic acid can also include synthetic or modified nucleic acids.

[0041] In some embodiments, the reference nucleic acid is obtained from a cell line suitable for use as a positive control for detecting one or more microdeletions.

[0042] In some embodiments, the reference nucleic acid is a mononucleosome.

[0043] In another aspect, the disclosure relates to a method for preparing a preparation of amplified DNA from a sample or a portion thereof useful for identifying one or more microdeletions associated with a disease or disorder, comprising: (a) preparing a construct for use as a positive control for detecting one or more microdeletions; (b) adding the construct from (a) to the sample or portion thereof to obtain a spiked sample; and extracting nucleic acid from the spiked sample or portion thereof; (c) performing targeted amplification on the spiked sample or portion thereof from (b) to amplify one or more target regions that include the microdeletions of interest to obtain amplicons; and (d) analyzing the amplicons or portions thereof from (c) to determine (i) whether the amplicons include the construct amplified as a positive control; and (ii) whether the amplicons include one or more microdeletions of interest.

[0044] In some embodiments, constructs for use as positive controls for the detection of one or more microdeletions are prepared by chemical synthesis followed by PCR amplification.

[0045] In some embodiments, the biological sample is a blood, plasma, serum, or urine sample. In some embodiments, the sample is a plasma sample and comprises cell-free DNA. In some embodiments, the sample comprises any fragment or segment of genomic DNA. In some embodiments, the sample comprises cellular DNA. As used herein, "cellular DNA" refers to DNA obtained from cells, organs, and tissues.

[0046] As used herein, the term "cell-free DNA" or "cfDNA" refers to DNA that is free-floating in a biological sample. In some embodiments, the biological sample is a blood, plasma, serum, or urine sample. In some embodiments, the sample is a plasma sample and contains cell-free DNA. In some embodiments, the biological sample is from a pregnant mother. In some embodiments, the isolated cfDNA is a mixture of fetal and maternal cfDNA. In some embodiments, the plasma sample contains maternal and fetal cell-free DNA, and the SNPs in the construct are altered to act as a positive control for fetal cell-free DNA.

[0047] In some embodiments, the biological sample contains circulating tumor DNA (ctDNA). In some embodiments, the compositions herein are used as a positive control for detecting ctDNA and detecting or monitoring cancer or tumors. ctDNA has been found in the circulation of patients diagnosed with malignant tumors, such as lung cancer, prostate cancer, colon cancer, and breast cancer. Identifying cancer-associated genomic instability, which can be determined in ctDNA from cancer patients, may be a diagnostic and prognostic tool. In one embodiment, the method of the present invention evaluates targeted microdeletions in a sample containing a mixture of nucleic acids from a subject suspected of or known to have cancer, such as carcinoma, sarcoma, lymphoma, leukemia, germ cell tumor, and blastoma. In one embodiment, the sample is a (processed) plasma sample derived from peripheral blood, containing a mixture of cfDNA (ctDNA) from normal cells and cancerous cells. In another embodiment, the biological sample required to determine whether a microdeletion is present is obtained from a mixture of cancerous and non-cancerous cells from other biological fluids including, but not limited to, serum, sweat, tears, sputum, urine, sputum, otorrhea, lymphatic fluid, saliva, cerebrospinal fluid, bone marrow suspension, vaginal flow, transcervical washings, cerebral fluid, ascites, milk, respiratory, intestinal and genitourinary secretions, and leukapheresis samples, or from a tissue biopsy, swab, or smear.

[0048] Chromosomal deletions involving tumor suppressor genes may play an important role in the development and progression of solid tumors. The retinoblastoma tumor suppressor gene (Rb-1), located on chromosome 13q14, is the most extensively characterized tumor suppressor gene. Altered or lost Rb protein expression is caused by inactivation of both gene alleles by point mutations or chromosomal deletions. Alterations in the Rb-1 gene have been found to be present not only in retinoblastoma but also in other malignancies, such as osteosarcoma, small cell lung cancer, and breast cancer. Restriction fragment length polymorphism (RFLP) studies have shown that loss of heterozygosity at 13q is frequent in these tumor types, suggesting that one of the Rb-1 alleles is lost due to a deletion of the entire chromosome. Abnormalities of chromosome 1, including duplications, deletions, and unbalanced translocations involving chromosome 6 and other partner chromosomes, suggest that regions of chromosome 1, particularly 1q21-1q32 and 1p11-13, may harbor oncogenes or tumor suppressor genes that are etiologically relevant to both chronic and advanced myeloproliferative neoplasms. Myeloproliferative neoplasms are also associated with deletions of chromosome 5. Complete or interstitial deletions of chromosome 5 are the most common karyotypic abnormality in myelodysplastic syndromes (MDS). Patients with isolated del(5q) / 5q-MDS have a better prognosis than patients with additional karyotypic defects, who are prone to developing myeloproliferative neoplasms (MPNs) and acute myeloid leukemia. Further cancer-associated microdeletion candidates may include the ribosomal subunit RPS14, the transcription factor Egr1 / Krox20, and the cytoskeletal remodeling protein α-catenin. Cytogenetic and allelotyping studies of fresh tumors and tumor cell lines have shown that allelic deletions from several different regions on chromosome 3p, including 3p25, 3p21-22, 3p21.3, 3p12-13, and 3p14, are the earliest and most frequent genomic abnormalities involved in a wide range of primary epithelial cancers of the lung, breast, kidney, head and neck, ovary, cervix, colon, pancreas, esophagus, bladder, and other organs.Several tumor suppressor genes have been mapped to the chromosome 3p region, and it is believed that stromal deletion or promoter hypermethylation precedes the loss of 3p or the entire chromosome 3 in cancer development.

[0049] Newborns and children with Down syndrome (DS) frequently suffer from congenital transient leukemia and are at increased risk for acute myeloid leukemia and acute lymphoblastic leukemia. Chromosome 21, which contains approximately 300 genes, may be involved in numerous structural abnormalities, such as translocations, deletions, and amplifications, in leukemia, lymphoma, and solid tumors. Furthermore, genes located on chromosome 21 that play important roles in tumorigenesis have been identified. Numerical and structural abnormalities of somatic chromosome 21 have been associated with leukemia, and specific genes located on 21q, such as RUNX1, TMPRSS2, and TFF, are involved in tumorigenesis.

[0050] In some embodiments, at least five microdeletions of interest are amplified in a single reaction volume, and a construct for use as a positive control is prepared for each of the at least five microdeletions of interest. In some embodiments, methods for preparing a preparation of amplified DNA from a sample or a portion thereof useful for identifying one or more microdeletions associated with a disease or disorder disclosed herein include a 22q11.2 deletion (DiGeorge syndrome), a chromosome 5p15.2 (Cri-du-Chat syndrome), a 1p36 deletion, a 15q11.2-q13 deletion (Prader-Willi syndrome), and / or a 15q11-q13 (Angelman syndrome).

[0051] Further examples of microdeletion regions of interest include one or more of the regions associated with the following genetic conditions and diseases: 1q21.1 distal microdeletion, 2q37 microdeletion: Albright hereditary osteodystrophy-like / brachydactyly, 3q29 microdeletion, Wolf-Hirschhorn syndrome, William-Beuren syndrome, Langer-Gideon / trichorhinophalangeal syndrome type 2, 9q34 microdeletion / Kleefstra syndrome, 10p13-p14 DiGeorge 2, 11p13 microdeletion: WAGR, 11q24.1 microdeletion: Jacobsen syndrome, Angelman syndrome type 2, Prader-Willi syndrome type 2, Prader-Willi, 16p11.2 microdeletion, 16pter-p13.3 microdeletion: AT-ID, Smith-Magenis, Miller-Dieker syndrome, RCAD (17q12 deletion), 17q21.31 microdeletion, 18q21.2 microdeletion Deletions: Pitt-Hopkins syndrome, DiGeorge, 22q11.21 microdeletion, 22q11.2 microdeletion, Phelan-McDermid 22q13 deletion, 5q22 microdeletion: familial adenomatous polyposis with ID, 5q35.2-35.3 microdeletion - Sotos syndrome, 6p25.3 (p24) microdeletion, 8p23.1 microdeletion CDH2, 11p11 .2 microdeletion: Potocki-Schaffer syndrome, 13q14.2 deletion, retinoblastoma with ID, 13q32 deletion-HPE5, PKD1 / TSC2 contiguous deletion syndrome, 17p13.3 distal microdeletion, 17p13.3 distal microdeletion, 17q21.31 microdeletion, isochromosome, 21q22.3 microdeletion: holoprosencephaly 1, Pelizaeus-Merzbach XL.

[0052] In some embodiments, the microdeletion of interest may be associated with cancer.

[0053] In some embodiments, the methods disclosed herein for preparing a preparation of amplified DNA from a sample or a portion thereof useful for identifying one or more microdeletions associated with a disease or disorder further comprise sequencing to detect (i) the presence of a construct as a positive control, and (ii) the presence of one or more microdeletions of interest.

[0054] In some embodiments, the methods disclosed herein for preparing a preparation of amplified DNA from a sample or portion thereof useful for identifying one or more microdeletions associated with a disease or disorder use positive controls to determine the efficiency and error rate of each amplification reaction, and use the efficiency and error rate to determine the presence of one or more microdeletions of interest.

[0055] Microdeletions of interest can be identified by combining the positive control constructs disclosed herein with the methods for identifying microdeletions described in U.S. Patent No. 17 / 252,205, which is incorporated by reference in its entirety.

[0056] In some embodiments, the present disclosure provides that the amount of construct added to a sample is determined by: (a) mixing the construct with mononucleosomal DNA from a normal female cell line in a range of ratios to generate a titration series to determine the limit of detection; (b) adding the mixture of (a) to DNA-depleted plasma; (c) performing targeted amplification of a microdeletion in which the construct is a positive control; and (d) determining the ratio of the construct to mononucleosomal DNA from the normal cell line that allows detection of the construct.

[0057] Some embodiments of the invention are kits that include compositions comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, or 50 different engineered nucleic acid constructs, and at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 50, 100, or 250 different engineered nucleic acid constructs, where each construct is a positive control for the microdeletion region of interest. In some embodiments, the kit may include a composition comprising: (i) a first engineered nucleic acid construct comprising a barcode replacing a targeted microdeletion corresponding to a 22q11.2 deletion, (ii) a second engineered nucleic acid construct comprising a barcode replacing a targeted microdeletion corresponding to a 5p15.2 deletion, (iii) a third engineered nucleic acid construct comprising a barcode replacing a targeted microdeletion corresponding to a 1p36 deletion, (iv) a fourth engineered nucleic acid construct comprising a barcode replacing a targeted microdeletion corresponding to a 15q11.2-q13 deletion, and / or (v) a fifth engineered nucleic acid construct comprising a barcode replacing a targeted microdeletion corresponding to a 15q11-q13 deletion. The kit may include primers for amplifying one or more targeted microdeletions and corresponding engineered nucleic acid constructs used as positive controls.

[0058] Nucleic acid isolation As used herein, the term "isolated" means that the target genetic material is physically separated from other biological material, and may also refer to partial isolation, where the target is isolated from some or most, but not all, of the biological material.

[0059] Isolation of mononucleosomal DNA: cfDNA has been shown to exist as nucleosomal complexes, in which DNA is tightly wrapped around histones. Mononucleosomal complexes consist of approximately 130 to 170 bp of DNA wrapped around a single nucleosome. The term "mononucleosome" refers to a fragment of chromosomal DNA containing a single nucleosome or a nucleic acid sequence containing the mononucleosomal DNA size of approximately 130 to 170 bp. The term "submononucleosome" refers to a fragment of chromosomal DNA with a molecular size smaller than approximately 130 bp, which is expected to be derived from a complete nucleosome. cfDNA may also exist incorporated into lipid vesicles such as exosomes.

[0060] Chromosomal DNA consists of DNA wrapped around complexes of histone proteins that form nucleosomes, which protect DNA, and fragmented chromosomal DNA is often found as multiple nucleosomes.

[0061] Cell-free DNA can be isolated from biological samples using many methods known to those skilled in the art. These methods include, but are not limited to, organic-liquid phase extraction, which utilizes phenol and phenol-chloroform mixtures to break down nucleoprotein complexes and sequester proteins and lipids in the organic phase, while partitioning highly hydrophilic DNA and RNA into the aqueous phase in highly purified form. Other methods include the use of agarose hydrogels, as described by E.M. Southern (J. Mol. Biol. (1975) 94:51-70) and Vogelstein and Gillespie (PNAS, USA (1979) 76:615-619), both of which are incorporated herein in their entireties. Another method involves capturing DNA on a solid-phase material, as described by Boom et al. (J. Clin. Micro. (1990) 28(3):495-503), both of which are incorporated herein in their entireties. General DNA isolation methods are described in Sambrook J, Russell DW (2001). Molecular Cloning: A Laboratory Manual 3rd Ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY, incorporated herein.

[0062] In certain embodiments, cfDNA is extracted by using an ion-exchange-based bead method.

[0063] Additional methods, described in detail below, can be used to enrich for DNA fragments within a particular molecular size range.

[0064] Size selection / exclusion method The present disclosure relates to methods that involve performing size selection by gel electrophoresis, paramagnetic beads, spin columns, salt precipitation, or biased amplification.

[0065] In some embodiments, the size exclusion step of the methods disclosed herein is performed by isolating a cfDNA sample according to size using gel electrophoresis and selecting a determined size range. Gel electrophoresis is an art-recognized method for isolating DNA molecules based on size by applying an electric field to a gel, such as an agarose gel, on which the DNA molecules migrate through the gel toward a positively charged anode. The size of the DNA molecule determines the speed at which the DNA molecule migrates through the gel. A standard mixture of DNA molecules of a predetermined size can be applied to the gel to identify the size of the DNA. DNA molecules of the desired size can then be extracted and purified using well-known techniques, such as those disclosed in Sambrook J, Russell DW (2001). Molecular Cloning: A Laboratory Manual 3rd Ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY. In some embodiments, size selection is performed using an automated high-throughput gel electrophoresis system, such as a Pippin or Costal Genomics system.

[0066] In some embodiments, the size exclusion step of the methods disclosed herein is carried out using paramagnetic beads. The use of paramagnetic beads for size selection of DNA fragments is described in DeAngelis et al., Solid-Phase Reversible Immobilization for the Isolation of PCR Products, Nucleic Acid Research, November 23(22):4742-3 (1995), incorporated herein by reference. Briefly, this method is based on the idea that the size of DNA fragments affects the total charge per molecule, with larger DNA fragments carrying a higher charge, promoting electrostatic interactions with the beads and displacing smaller fragments. Therefore, by manipulating the composition of the buffer used to mix the beads and DNA, beads can be made to bind DNA within a specific size range. The most well-known and widely applicable approach is solid-phase reversible isolation (SPRI) selection. This utilizes carboxyl-coated paramagnetic beads in the presence of a high concentration of salt and the crowding agent polyethylene glycol (PEG), which promotes controlled adsorption and is configured to bind DNA molecules within a specific molecular weight range by varying the PEG concentration. DNA molecules of different lengths can be resolved by subjecting the source DNA to various binding and elution schemes in the presence of different amounts of PEG. In some embodiments, AMPURE™ beads are used for the size exclusion step.

[0067] In some embodiments, the size exclusion step of the methods disclosed herein is performed using a spin column. The spin column contains a material that absorbs molecules based on their size. The spin column material contains pores of defined sizes, and molecules with sizes above a cutoff size determined by the pore size do not enter the pores and are eluted in the void volume of the column. Various types of column materials can be selected to achieve absorption or exclusion of DNA molecules of various size ranges. In some embodiments, the spin column material comprises a siliceous material, silica gel, glass, glass fiber, zeolite, aluminum oxide, titanium dioxide, zirconium dioxide, kaolin, gelatinous silica, magnetic particles, ceramic, polymer support material, or a combination thereof. In certain embodiments, the spin column material comprises glass fiber.

[0068] In some embodiments, spin columns may be used for size exclusion by using different binding buffers configured to provide low or high stringency binding conditions when applying a DNA sample to the spin column, as described in PCT Patent Application No. PCT / US2019 / 18274, filed February 15, 2019, which is incorporated herein by reference in its entirety. Under low stringency binding conditions, the spin column material is configured to restrict binding of low molecular weight DNA fragments, while under high stringency binding conditions, the spin column is configured to promote binding of low molecular weight DNA fragments.

[0069] In some embodiments, the low stringency and / or high stringency binding buffer comprises a nitrile compound selected from acetonitrile (ACN), propionitrile (PCN), butyronitrile (BCN), isobutylnitrile (IBCN), or a combination thereof. The first and / or second binding buffer may comprise, for example, about 15% to about 35%, or about 20% to about 30%, or about 25% of a nitrile compound (e.g., ACN).

[0070] In some embodiments, the low stringency and / or high stringency binding buffer comprises a chaotropic compound selected from GnCl, urea, thiourea, guanidine thiocyanate, NaI, guanidine isothiocyanate, D- / L-arginine, perchlorate or perchlorate salts of Li, Na, K, or combinations thereof. The low stringency and / or high stringency binding buffer can comprise, for example, about 5 M to about 8 M, or about 5.6 M to about 7.2 M, or about 6 M of a chaotropic compound (e.g., GnCl).

[0071] The binding buffer may also contain an alcohol, a chelating agent, and a surfactant. In some embodiments, the alcohol is propanol. In some embodiments, the chelating compound comprises ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), citric acid, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), 2,2'-bipyridyl, deferoxamine methanesulfonate (DFOM), 2,3-dihydroxybutanedioic acid (tartaric acid), or a combination thereof. In some embodiments, the surfactant may be Triton X-100, Tween 20, N-lauroylsarcosine, sodium dodecyl sulfate (SDS), dodecyldimethylphosphine oxide, sorbitan monopalmitate, decylhexaglycol, 4-nonylphenyl-polyethylene glycol, or a combination thereof. In certain embodiments, the surfactant is Triton X-100.

[0072] In some embodiments, the size exclusion step of the methods disclosed herein is carried out using salt precipitation. Larger DNA molecules precipitate at lower salt concentrations than smaller DNA molecules. By varying the salt concentration in the precipitation buffer, DNA molecules of different size ranges can be isolated.

[0073] Amplification Method In some embodiments, the method includes performing targeted amplification to identify microdeletions of interest. In some embodiments, the targeted amplification to identify microdeletions of interest includes performing amplification in multiple amplification reactions. In some embodiments, the amplification reactions are single reactions performed in parallel. In some embodiments, the target loci are amplified by performing multiplex amplification of multiple target loci in a single reaction. In some embodiments, the amplification reactions to identify microdeletions of interest are performed by using multiplex amplification of multiple target loci in a single reaction. In some embodiments, at least five microdeletions of interest are identified by performing multiplex amplification in a single reaction volume.

[0074] In some embodiments, the reaction mixture for performing the multiplex amplification includes an engineered construct for use as a positive control for identifying the microdeletion of interest.

[0075] The multiplex reaction can be set up as a single reaction or as a pool of different subsets of multiplex reactions. The multiplex reaction methods provided herein, such as the massively multiplexed PCR disclosed herein, provide exemplary processes for performing amplification reactions to help achieve improved multiplexing and, therefore, sensitivity levels.

[0076] In certain illustrative embodiments, the nucleic acid sequence data is generated by performing high-throughput DNA sequencing of multiple copies of a series of amplicons generated using a multiplex amplification reaction, where each amplicon in the series spans at least one polymorphic locus of a set of polymorphic loci, and each of the polymorphic loci in the set is amplified. For example, in these embodiments, multiplex PCR may be performed to amplify amplicons across five microdeletion regions, where the amplicons comprise multiple polymorphic loci.

[0077] In some embodiments, the amplification is performed using direct multiplex PCR, sequential PCR, nested PCR, double nested PCR, one-and-a-half PCR, or any combination thereof. sided nested PCR, fully nested PCR, one-sided fully nested PCR, one-sided nested PCR, heminested PCR, heminested PCR, triplex heminested PCR, semi-nested PCR, one-sided semi-nested PCR, reverse semi-nested PCR, or one-sided PCR, as described in U.S. Application No. 13 / 683,604 filed November 21, 2012, U.S. Publication No. 2013 / 0123120, U.S. Application No. 13 / 300,235 filed November 18, 2011, U.S. Publication No. 2012 / 0270212, and U.S. Application No. 61 / 994,791 filed May 16, 2014, which are incorporated by reference in their entireties.

[0078] In one embodiment, the multiplex PCR assay is designed to amplify the microdeletion region of interest and / or amplify SNPs within or flanking the microdeletion of interest.

[0079] In some embodiments, a method for amplifying target loci in a nucleic acid sample includes (i) contacting the nucleic acid sample with a library of primers that simultaneously hybridize to at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 750, 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different target loci to generate a single reaction mixture, and (ii) subjecting the reaction mixture to primer extension reaction conditions (such as PCR conditions) to generate amplified products comprising the 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. In some embodiments, the primers are in solution (e.g., dissolved in a liquid phase rather than a solid phase). In some embodiments, the primers are in solution and not immobilized on a solid support. In some embodiments, the primers are not part of a microarray.

[0080] In certain embodiments, the multiplex amplification reaction is performed under limiting primer conditions for at least half of the reactions. In some embodiments, limiting primer concentrations are used in 1 / 10, 1 / 5, 1 / 4, 1 / 3, 1 / 2, or all of the reactions in the multiplex reaction. Factors to consider in achieving limiting primer conditions in amplification reactions such as PCR are provided herein.

[0081] The PCR reaction conditions may be, for example, the STAR (target specific amplification reaction) protocol disclosed in U.S. Patent No. 17 / 545,881, which is incorporated herein in its entirety. For example, the PCR reaction may be carried out at an annealing temperature of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10° C. above the melting point at the lower end of the range, and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15° C. above the melting point at the upper end of the range, for at least 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 95, or 100% of the primers in the set of primers.

[0082] In certain embodiments, when the amplification reaction is a PCR reaction, the length of the annealing step in the PCR reaction is 1, 10, 15, 20, 30, 45, and 60 minutes at the lower end of the range, and 15, 20, 30, 45, 60, 120, 180, or 240 minutes at the higher end of the range. In certain embodiments, the primer concentration in the amplification, such as a PCR reaction, is 1 to 10 nM. Furthermore, in exemplary embodiments, the primers in the primer set are designed to minimize primer-dimer formation.

[0083] Thus, in one example of any of the methods herein that include an amplification step, the amplification reaction is a PCR reaction, the annealing temperature is 1-10°C higher than the melting temperature of at least 90% of the primers in the primer set, the length of the annealing step in the PCR reaction is 15-60 minutes, the primer concentration in the amplification reaction is 1-10 nM, and the primers in the primer set are designed to minimize primer-dimer formation. In a further aspect of this example, the multiplex amplification reaction is performed under limiting primer conditions.

[0084] In some embodiments, the following ranges of multiplex reactions are performed: At the lower end of the range, between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 500, 1,000, 2,500, 5,000, 10,000, 20,000, 25,000, 50,000, and at the upper end of the range, between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 500, 1,000, 2,500, 5,000, 10,000, 20,000, 25,000, 50,000, and 100,000.

[0085] In one embodiment, multiplex PCR assays are designed to amplify SNPs within or on either side of the microdeletion of interest, and these assays are used in a single reaction to amplify DNA. The number of PCR assays can be 1-10, 5-10, 10-50, 50-200, 5-20, 100-1,000, 1,000-10,000, 1,000-20,000, or even 20,000 or more PCR assays (5-20-plex, 100-1,000-plex, 1,000-5,000-plex, 1,000-10,000-plex, 1,000-20,000-plex, or 20,000-plex or more, respectively). In one embodiment, a multiplex pool of approximately 10,000 PCR assays (10,000-plex) is designed to amplify target loci of interest, and these assays are used in a single reaction to amplify cfDNA obtained from plasma samples, chorionic villus samples, amniocentesis samples, single or small numbers of cells, other bodily fluids or tissues, cancers, or genetic material from sources.

[0086] The SNP frequency of each locus can be determined by clonal methods or some other method of sequencing the amplicon. Statistical analysis of the allele frequency distribution or the ratio of all assays can be used to determine whether a sample contains one or more trisomies of the chromosomes included in the test. In another embodiment, the original cfDNA sample is split into two samples and parallel 5,000-plex assays are performed. In another embodiment, the original cfDNA sample is split into n samples and parallel (approximately 10,000 / n)-plex assays are performed, where n is 2 to 12, or 12 to 24, or 24 to 48, or 48 to 96.

[0087] Bioinformatics methods are used to analyze the genetic data obtained from the multiplex PCR. Bioinformatics methods useful and relevant to the methods disclosed herein can be found in U.S. Patent Publication No. 20180025109, which is incorporated herein by reference.

[0088] Hybrid Capture Method In some embodiments, the method includes performing hybrid capture to select a plurality of polymorphic loci on the selectively enriched DNA prior to sequencing the selectively enriched DNA.

[0089] In some embodiments, preferentially enriching DNA at a plurality of polymorphic loci includes obtaining a plurality of hybrid capture probes that target the polymorphic loci, hybridizing the hybrid capture probes to DNA in the sample, and physically removing some or all of the unhybridized DNA from the first DNA sample from the sample.

[0090] In some embodiments, the hybrid capture probes are designed to hybridize to regions on either side of the polymorphic site but not overlapping. In some embodiments, the hybrid capture probes are designed to hybridize to regions on either side of the polymorphic site but not overlapping, and the length of the capture probes on either side can be selected from the group consisting of less than about 120 bases, less than about 110 bases, less than about 100 bases, less than about 90 bases, less than about 80 bases, less than about 70 bases, less than about 60 bases, less than about 50 bases, less than about 40 bases, less than about 30 bases, and less than about 25 bases. In some embodiments, the hybrid capture probes are designed to hybridize to regions that overlap the polymorphic site, and the plurality of hybrid capture probes includes at least two hybrid capture probes for each polymorphic locus, each hybrid capture probe designed to be complementary to a different allele at the polymorphic locus.

[0091] Nucleic acid sequencing The sample nucleic acid can be further analyzed by microarray genotyping and high-throughput sequencing. Some high-throughput sequencing methods include Sanger DNA sequencing, pyrosequencing, the ILLUMINA SOLEXA platform, the ILLUMINA genome analyzer, or the APPLIED BIOSYSTEM 454 sequencing platform, the HELICOS TRUE SINGLE MOLECULE SEQUENCING platform, the HALCYON MOLECULAR electron microscope sequencing, or any other sequencing method. In some embodiments, high-throughput sequencing is performed on an Illumina NextSeq. In some embodiments, high-throughput sequencing methods include NovaSeq, BGI / MGI sequencing system, Omniome / PacBio sequencing system, Oxford Nanopore sequencing system, or Ion Torrent sequencing system.

[0092] In some embodiments, the sequence of the selectively enriched DNA is determined by performing microarray analysis. In one embodiment, the microarray may be an ILLUMINA SNP microarray or an AFFYMETRIX SNP microarray.

[0093] In some embodiments, the sequence is determined by quantitative PCR (qPCR) or digital droplet PCR (ddPCR) analysis. qPCR measures the intensity of fluorescence at a specific time (generally after each amplification cycle) to determine the relative amount of target molecules (DNA). ddPCR measures the actual number of molecules (target DNA) because each molecule is in a droplet, thus making it a separate "digital" measurement. This provides absolute quantification because ddPCR measures the positive fraction of the sample, i.e., the number of droplets that fluoresce due to proper amplification. This positive fraction accurately indicates the initial amount of template nucleic acid.

[0094] As used herein, the term "adapter," or "ligation adapter," or "library tag," refers to a DNA molecule containing a universal priming sequence that can be covalently attached to the 5-prime and 3-prime ends of a population of target double-stranded DNA molecules. In some embodiments, the addition of the adapter provides universal priming sequences at the 5-prime and 3-prime ends of the target population, allowing PCR amplification to be performed, and a single amplification primer pair is used to amplify all molecules from the target population. Disclosed herein are methods that enable targeted amplification across hundreds to thousands of target sequences (e.g., SNP loci) from genomic DNA obtained from plasma. Amplified samples are relatively free of primer-dimer products and may have low allelic bias at target loci. If sequencing-compatible adapters are added to the products during or after amplification, analysis of these products can be performed by sequencing. These methods are described in further detail in U.S. Patent Publication Nos. 20170242960 and 20180025109, and U.S. Patent No. 9,163,282, which are incorporated herein.

[0095] In some embodiments, the adapters or primers described herein may include one or more molecular barcodes. Molecular barcodes or molecular index sequences may be used in next-generation sequencing to mitigate quantitative bias caused by duplication. In next-generation sequencing, each nucleic acid fragment may be tagged with a molecular barcode or molecular index sequence. Sequence reads with different molecular barcodes or molecular index sequences represent different original nucleic acid molecules. By referencing the molecular barcodes or molecular index sequences, PCR artifacts, such as sequence changes generated by polymerase errors that are not present in the original nucleic acid molecule, can be distinguished and separated from actual variants / mutations present in the original nucleic acid molecule.

[0096] In some embodiments, the molecular barcodes are introduced by ligating adapters having the molecular barcodes to the isolated cfDNA, resulting in adapter-ligated, molecularly barcoded DNA. In some embodiments, the molecular barcodes are introduced by amplifying the adapter-ligated DNA with primers having the molecular barcodes, resulting in amplified adapter-ligated, molecularly barcoded DNA.

[0097] In some embodiments, a molecular barcode adapter or primer may contain a universal sequence followed by a molecular barcode region, and optionally, in the case of a primer, followed by a target-specific sequence. The 5' sequence of the molecular barcode can be used for subsequence PCR amplification or sequencing and may contain sequences useful for converting amplicons into a library for sequencing. Random molecular barcode sequences may be generated in a variety of ways. A preferred method is to synthesize molecular tagging adapters or primers such that all four bases are included in the reaction during synthesis of the barcode region. All or various combinations of bases can be specified using IUPAC DNA ambiguity codes. In this way, the collection of molecules synthesized will contain a random mix of sequences within the molecular barcode region. The length of the barcode region determines the number of adapters or primers that contain unique barcodes. The number of unique sequences is determined by N L The length of the barcode region is related to N as where N is the number of bases (usually 4) and L is the length of the barcode. A 5-base barcode can generate up to 1024 unique sequences. An 8-base barcode results in 65536 unique barcodes. In one embodiment, DNA can be measured by a sequencing method where the sequence data represents the sequence of a single molecule. This can include methods where single molecules are directly sequenced or where single molecules are amplified to form clones that are detectable by sequencing equipment but still represent single molecules (referred to herein as clonal sequencing).

[0098] In some embodiments, the molecular barcodes described herein are molecular index tags ("MITs") that are attached to a population of nucleic acid molecules from a sample and identify individual sample nucleic acid molecules from the population of nucleic acid molecules (i.e., members of the nucleic acid molecules) after sample processing for a sequencing reaction. MITs are described in detail in U.S. Patent No. 10,011,870 to Zimmermann et al., which is incorporated herein by reference in its entirety. Unlike prior art methods that teach having a diversity of unique identifiers greater than the number of sample nucleic acid molecules in a sample in order to associate and tag each sample nucleic acid molecule with a unique identifier, the present disclosure typically involves sample nucleic acid molecules that are much greater than the diversity of MITs in the set of MITs. Indeed, the methods and compositions herein provide for the identification of 1,000, 1x10, or 1x10 distinct MITs for each distinct MIT in the set of MITs. 6 pieces, 1×10 9 The method may include individual or even more starting molecules, and still be able to identify the individual sample nucleic acid molecules that give rise to tagged nucleic acid molecules after amplification.

[0099] General definition As used in the description of this invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0100] As used herein, the term "about," when referring to a measurable value such as an amount or concentration, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0101] When used to describe the selection of any component, range, dosage form, etc. disclosed herein, the terms "acceptable," "effective," or "sufficient" intend that the component, range, dosage form, etc. is suitable for the purposes of the disclosure.

[0102] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0103] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) is intended to be interpreted as including the recited materials or steps and "that do not materially affect the basic and novel characteristic(s)" of the recited embodiment. See In re Herz, 537 F.2d 549,551-52,190 USPQ 461,463 (CCPA 1976) (emphasis in original). See also MPEP § 2111.03. Thus, as used herein, the term "consisting essentially of" should not be construed as equivalent to "comprising." "Consisting of" is intended to mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions disclosed herein. Embodiments defined by each of these transitional terms are within the scope of this disclosure. [Example]

[0104] Example 1 This example demonstrates the construction of an engineered construct for use as a positive control in a method for identifying microdeletions of interest, and the use of the construct to identify microdeletions of interest.

[0105] The overall method for constructing an engineered construct for use as a positive control in the method for identifying microdeletions of interest is shown in Figure 1. First, a reference DNA sequence containing the microdeletion region of interest is obtained. In Figure 1, the "^" symbol indicates a single nucleotide polymorphism (SNP), and "v" indicates an inversion SNP. Next, a region within the microdeletion region is deleted and replaced with a 6- to 9-base pair barcode. Regions in the 5' and 3' regions on either side of the central microdeletion region contain primer binding sites suitable for use with STAR (specific target amplification reaction) primers targeting the microdeletion. Primers can be designed for identification of 22q11.2 deletions (DiGeorge syndrome), chromosome 5p15.2 (Cri-du-Chat syndrome), 1p36 deletions, 15q11.2-q13 deletions (Prader-Willi syndrome), and / or 15q11-q13 (Angelman syndrome).

[0106] The constructs are prepared using chemical synthesis followed by PCR amplification of the synthesized construct.

[0107] A construct engineered for use as a positive control is mixed with a nucleic acid sample from a subject to obtain a spiked sample. The amount of construct added to the sample is determined by (a) mixing the construct with mononucleosomal DNA from a normal female cell line in a range of ratios to generate a titration series to determine the limit of detection, (b) adding the mixture from (a) to DNA-depleted plasma, (c) performing targeted amplification of the microdeletion for which the construct is a positive control, and (d) determining the ratio of the construct to mononucleosomal DNA from the normal cell line that allows detection of the construct. The identified amount of construct is added to the sample from the subject, and targeted amplification of the microdeletion of interest is performed. The construct is amplified as a positive control and can be identified by a barcode that replaces the microdeletion region. Microdeletions of interest can be amplified in a single reaction volume, in which case a composition containing positive control constructs for all microdeletions of interest is added to the sample. Amplification of a positive control construct allows for determination of efficiency and error rate, which can be used to calibrate amplification results of sample nucleic acids and improve accuracy in identifying microdeletions of interest.

[0108] The demonstration that SNP assays on either side of the barcode replacing the microdeletion in the engineered construct work in a STAR reaction indicates that assays on either side of the deletion and within the deletion work, and therefore the construct can be used as a positive control.

Claims

1. 1. A composition comprising an engineered nucleic acid construct for use as a positive control for one or more primers targeting one or more microdeletions of interest in a sample, wherein the construct is engineered from a reference nucleic acid sequence comprising the microdeletion of interest, the construct comprising a 5' terminal region, a central region, and a 3' terminal region, the 5' terminal region and the 3' terminal region comprising reference sequences on either side of the microdeletion of interest, the central region of the construct being a DNA barcode, and the DNA barcode replacing the microdeletion sequence of the reference nucleic acid sequence.

2. 2. The composition of claim 1, wherein the microdeletion of interest corresponds to a 22q11.2 deletion, a 5p15.2 deletion, a 1p36 deletion, a 15q11.2-q13 deletion, or a 15q11-q13 deletion.

3. The composition comprises: (i) a first engineered nucleic acid construct comprising a barcode that replaces a targeted microdeletion corresponding to a 22q11.2 deletion; (ii) a 5 10. The composition of claim 1, comprising: (i) a second engineered nucleic acid construct comprising a barcode that replaces a targeted microdeletion corresponding to a p15.2 deletion; (iii) a third engineered nucleic acid construct comprising a barcode that replaces a targeted microdeletion corresponding to a 1p36 deletion; (iv) a fourth engineered nucleic acid construct comprising a barcode that replaces a targeted microdeletion corresponding to a 15q11.2-q13 deletion; and (v) a fifth engineered nucleic acid construct comprising a barcode that replaces a targeted microdeletion corresponding to a 15q11-q13 deletion.

4. The composition of claim 1 , wherein the targeted microdeletion is associated with cancer.

5. 10. The composition of claim 1, wherein the barcode is about 6 to about 9 base pairs.

6. The composition of claim 1 , wherein the 5′ terminal region and the 3′ terminal region of the construct comprise at least one single nucleotide polymorphism (SNP) of interest.

7. 2. The composition of claim 1, wherein the 5' and 3' terminal regions of the construct comprise sequences recognized by primers targeting SNPs within or on either side of the microdeletion of interest.

8. 4. The composition of claim 3, wherein the reference nucleic acid sequence is maternal DNA and the SNP of interest is altered so that the construct can act as a positive control for offspring DNA.

9. 2. The composition of claim 1, wherein the size of the construct is from about 100 bp to about 200 bp, or from about 160 bp to about 200 bp.

10. 10. A method for preparing a construct for use as a positive control for one or more primers targeting one or more microdeletions of interest in a sample according to claims 1 to 9, comprising obtaining a reference nucleic acid, isolating a nucleic acid sequence comprising the 5' and 3' ends on either side of the microdeletion of interest, and replacing the central region of the reference nucleic acid sequence corresponding to the microdeletion of interest with a barcode.

11. 11. The method of claim 10, wherein said preparing a construct for use as a positive control for one or more primers targeting one or more microdeletions is performed by chemical synthesis of said construct and subsequent PCR amplification of the synthesized construct.

12. 11. The method of claim 10, wherein the sample is a plasma sample and comprises cell-free DNA.

13. 11. The method of claim 10, wherein the sample comprises circulating tumor DNA (ctDNA).

14. The method of claim 10, wherein the sample comprises cells and / or tissues.

15. 11. The method of claim 10, wherein the reference nucleic acid is obtained from a cell line suitable for use as a positive control for detecting the one or more microdeletions.

16. The method of claim 10, wherein the reference nucleic acid is genomic DNA.

17. The method of claim 10, wherein the reference nucleic acid is a mononucleosome.

18. 1. A method for preparing a preparation of amplified DNA from a sample or a portion thereof useful for identifying one or more microdeletions associated with a disease or disorder, comprising: (a) preparing a construct for use as a positive control for detecting one or more microdeletions according to claims 10 to 17; (b) adding the construct from (a) to the sample or a portion thereof to obtain a spiked sample, and extracting nucleic acid from the spiked sample or a portion thereof; (c) performing targeted amplification on the spiked sample from (b) or a portion thereof to amplify one or more target regions containing the microdeletion of interest to obtain amplicons; and (d) analyzing the amplicon or a portion thereof from (c) to determine (i) whether the amplicon contains the amplified construct as a positive control, and (ii) whether the amplicon contains one or more microdeletions of interest.

19. 20. The method of claim 18, wherein the sample is a plasma sample and comprises cell-free DNA.

20. 20. The method of claim 19, wherein the plasma sample contains maternal and fetal cell-free DNA, and the SNP in the construct is altered to act as a positive control for the fetal cell-free DNA.

21. 21. The method of claim 20, wherein at least five microdeletions of interest are amplified in a single reaction volume, and a construct for use as a positive control is prepared for each of the at least five microdeletions of interest.

22. 22. The method of claims 18 to 21, wherein the one or more microdeletions comprise a 22q11.2 deletion (DiGeorge syndrome), chromosome 5p15.2 (Cri-du-Chat syndrome), a 1p36 deletion, a 15q11.2-q13 deletion (Prader-Willi syndrome), and / or a 15q11-q13 (Angelman syndrome).

23. The method of claims 18 to 21, wherein the one or more microdeletions are associated with cancer.

24. 19. The method of claim 18, further comprising sequencing to detect (i) the presence of the construct as a positive control, and (ii) the presence of the one or more microdeletions of interest.

25. 19. The method of claim 18, wherein an efficiency and error rate are determined for each amplification reaction using the positive control, and the efficiency and error rate are used to determine the presence of the one or more microdeletions of interest.

26. 19. The method of claim 18, wherein the amount of the construct added to the sample is determined by: (a) mixing the construct with DNA from a normal female cell line in a range of ratios to generate a titration series to determine the limit of detection; (b) adding the mixture of (a) to DNA-depleted plasma; (c) performing targeted amplification of the microdeletion in which the construct is a positive control; and (d) determining the ratio of the construct to mononucleosomal DNA from the normal cell line that allows detection of the construct.

27. 27. The method of claim 26, wherein the DNA is mononucleosomal DNA.

28. 19. The method of claim 18, wherein the sample is a plasma sample from the mother.

29. A method for preparing a sample containing nucleic acids, comprising spiking the composition of claims 1 to 9 into the sample.

30. 30. The method of claim 29, wherein the sample is a plasma sample from the mother.

31. 31. The method of any one of claims 18 to 30, wherein detection of SNPs in the amplicon flanking the barcode that replaces the microdeletion in the engineered construct demonstrates that the assay works to detect SNPs flanking and within the microdeletion, thereby confirming that the engineered construct can be used as a positive control.

Images