Compositions and methods for identifying chromosomal microdeletions

Abstract

Provided herein are compositions for use as positive controls for detecting one or more microdeletions of interest in a sample. The positive controls can be used to determine the error and efficiency of an assay for identifying microdeletions, such as a 22q11.2 deletion (Digille Syndrome), chromosome 5p15.2 (Cat Syndrome), 1p36 deletion, 15q11.2-q13 deletion (Prallard-Wili Syndrome), and / or 15q11-q13 (Angiel Syndrome).

Description

Background Art

[0001] Subchromosome abnormalities, such as microdeletions and duplications, can lead to severe physical and / or intellectual disability. The combined incidence of the eight microdeletion syndromes exceeds 1 in 1,000, making them almost as common as fetal autosomal trisomy.

[0002] In young women, the risk of developing a clinically significant microdeletion exceeds the risk of Down syndrome. Because some infants with subchromosomal abnormalities may benefit from early therapeutic intervention, prenatal testing is important to optimize treatment.

[0003] Subchromosomal abnormalities, such as microdeletions or duplications, are relatively more difficult to detect than chromosomal abnormalities due to their smaller size. There is a need for improved methods and compositions for detecting subchromosomal abnormalities, such as microdeletions. Summary of the invention

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

[0005] The present disclosure provides a composition comprising an engineered nucleic acid construct, the construct being used as a positive control for detecting 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, wherein the construct comprises a 5' terminal region, a central region, and a 3' terminal region, wherein the 5' terminal region and the 3' terminal region comprise reference sequences flanking the microdeletion of interest, wherein the central region of the construct is a DNA barcode, and wherein the barcode replaces 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 microdeletion of interest corresponding to a 22q11.2 deletion; (ii) a second engineered nucleic acid construct comprising a barcode that replaces a microdeletion of interest corresponding to a 5p15.2 deletion; (iii) a third engineered nucleic acid construct comprising a barcode that replaces a microdeletion of interest corresponding to a 1p36 deletion; (iv) a fourth engineered nucleic acid construct comprising a barcode that replaces a microdeletion of interest corresponding to a 15q11.2-q13 deletion; and (v) a fifth engineered nucleic acid construct comprising a barcode that replaces a microdeletion of interest 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 comprise sequences recognized by primers targeting SNPs within or flanking a microdeletion of interest.

[0011] In some embodiments, the reference nucleic acid sequence is maternal DNA, and wherein the SNP of interest is altered to allow the construct to serve as a positive control for progeny DNA.

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

[0013] 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, wherein the method comprises obtaining a reference nucleic acid; isolating nucleic acid sequences comprising the 5' end and the 3' end flanking 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 the one or more microdeletions.

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

[0016] In another aspect, the present disclosure relates to a method for preparing a preparation of amplified DNA derived from a sample or a fraction thereof that can be used to identify one or more microdeletions associated with a disease or condition, the method 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 a fraction thereof to obtain a spiked sample and extracting nucleic acid from the spiked sample or a fraction thereof; (c) performing targeted amplification on the spiked sample or a fraction thereof from (b) to amplify one or more target regions containing the microdeletions of interest to obtain amplicons; and (d) analyzing the amplicons or portions thereof from (c) to determine (i) whether the amplicon contains the amplified construct as a positive control, and (ii) whether the amplicon contains the one or more microdeletions of interest.

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

[0018] In some embodiments, the sample is a plasma sample and comprises free DNA. In some embodiments, the plasma sample comprises maternal and fetal free DNA, and wherein the SNP in the construct is changed to serve as a positive control for the fetal free DNA. In some embodiments, the sample comprises circulating tumor DNA (ctDNA).

[0019] In some embodiments, at least 5 microdeletions of interest are amplified in a single reaction volume, and a construct used as a positive control is prepared for each of the at least 5 microdeletions of interest. In some embodiments, disclosed herein is a method for preparing a preparation of amplified DNA derived from a sample or fraction thereof that can be used to identify one or more microdeletions associated with a disease or condition, and the one or more microdeletions include 22q11.2 deletion (DiGeorge syndrome), chromosome 5p15.2 (Cri-du-chat syndrome), 1p36 deletion, 15q11.2-q13 deletion (Prader-Willi syndrome) and / or 15q11-q13 (Angelman syndrome).

[0020] In some embodiments, disclosed herein is a method for preparing a preparation of amplified DNA derived from a sample or a fraction thereof that can be used to identify one or more microdeletions associated with a disease or condition, the method 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.

[0021] In some embodiments, disclosed herein is a method for preparing a preparation of amplified DNA derived from a sample or a fraction thereof that can be used to identify one or more microdeletions associated with a disease or condition, by determining the efficiency and error rate of each amplification reaction using the positive control, wherein the efficiency and the error rate are used to determine the presence of the one or more microdeletions of interest.

[0022] In some embodiments, the disclosure provides the amount of the construct to be added to the sample, the amount being determined by: (a) mixing DNA from a normal female cell line with the construct 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 a normal cell line that allows detection of the construct. In some embodiments, the DNA is mononucleosomal or genomic DNA.

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

[0024] In some embodiments, detection of SNPs flanking the barcode that replace the microdeletion in the engineered construct in the amplicon indicates that the assay for detecting SNPs flanking and within the microdeletion is functional, thereby confirming that the engineered construct can be used as the positive control. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a graphic depiction of an engineered nucleic acid construct used as a positive control for detecting microdeletions. MD is an abbreviation for microdeletion. STAR primers refer to the STAR PCR protocol described elsewhere herein. STAR is an abbreviation for specific target amplification reaction.

[0026] Figure 2 is a flow chart illustrating the use of the engineered nucleic acid constructs disclosed herein as positive controls for detecting microdeletions. DETAILED DESCRIPTION

[0027] Now will be referred to in detail some specific embodiments of the present invention that the inventors have contemplated for implementing the present invention. Some examples of these specific embodiments are shown in the accompanying drawings. Although the present invention has been described in conjunction with these specific embodiments, it should be understood that it is not intended to limit the present invention to the embodiments described. On the contrary, it is intended to cover the alternatives, modifications and equivalents that may be included.

[0028] The present disclosure provides improved methods for determining microdeletions or other subchromosomal abnormalities of interest. In some embodiments, the present disclosure provides methods for non-invasive prenatal testing (NIPT), specifically, by performing targeted amplification and using engineered positive control constructs corresponding to microdeletions of interest disclosed herein to identify microdeletions or other subchromosomal abnormalities in a fetus.

[0029] In particular, the present disclosure provides a composition comprising an engineered nucleic acid construct, which is used as a positive control for detecting 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, wherein the construct comprises a 5' terminal region, a central region, and a 3' terminal region, wherein the 5' terminal region and the 3' terminal region comprise reference sequences flanking the microdeletion of interest, and wherein the central region of the construct is a DNA barcode, wherein the barcode replaces the microdeletion sequence of the reference nucleic acid sequence.

[0030] The barcode is unique to the specific microdeletion region, so the PCR for the positive control for the specific microdeletion PCR assay can be determined and analyzed. The PCR for the positive control 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 detection.

[0031] Figure 1 Provided is a graphic depiction of an exemplary engineered nucleic acid construct that is used as a positive control for detecting one or more microdeletions of interest as disclosed herein. Figure 1 As shown, the engineered nucleic acid construct comprises a barcode that replaces the region corresponding to the microdeletion of interest and retains the 5' terminal region and the 3' terminal region flanking the region corresponding to the microdeletion of interest. 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.

[0032] 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.

[0033] 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 Cry-a-Cour syndrome.

[0034] In some embodiments, the composition comprises: (i) a first engineered nucleic acid construct comprising a barcode that replaces a microdeletion of interest corresponding to a 22q11.2 deletion; (ii) a second engineered nucleic acid construct comprising a barcode that replaces a microdeletion of interest corresponding to a 5p15.2 deletion; (iii) a third engineered nucleic acid construct comprising a barcode that replaces a microdeletion of interest corresponding to a 1p36 deletion; (iv) a fourth engineered nucleic acid construct comprising a barcode that replaces a microdeletion of interest corresponding to a 15q11.2-q13 deletion; and (v) a fifth engineered nucleic acid construct comprising a barcode that replaces a microdeletion of interest corresponding to a 15q11-q13 deletion. In some embodiments, the composition comprises a plurality of engineered nucleic acid constructs, each of which is a positive control for a microdeletion region of interest. The composition can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, or 50 different engineered nucleic acid constructs at the low end and 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 50, 100, or 250 different engineered nucleic acid constructs at the high end, wherein each construct is a positive control for the microdeletion region of interest.

[0035] In certain embodiments, the 5' and 3' terminal regions of the construct comprise at least one SNP of interest (SNP). In certain embodiments, the 5' and 3' terminal regions of the construct comprise sequences identified by primers of the SNP of the targeted microdeletion or flank. In certain embodiments, the reference nucleic acid sequence is maternal DNA, and the SNP of interest (or inversion) is wherein changed to allow the construct to serve as a positive control for progeny DNA.

[0036] 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 the term should not imply any limitation on the frequency of occurrence of each variant.

[0037] The term "sequence" refers to a DNA sequence or gene sequence. It can refer to the basic physical structure of a DNA molecule or strand in an individual. It can refer to the sequence of nucleotides found in a DNA molecule, or the complementary strand of a DNA molecule. It can refer to the information contained in a DNA molecule as its bioinformatics representation.

[0038] The term "locus" refers to a specific region of interest on an individual's DNA, which may refer to a SNP, a site of insertion or deletion, a chromosome or portion thereof, or a site of some other relevant genetic variation. A disease-associated SNP may also refer to a disease-associated locus.

[0039] The term "polymorphic allele" or "polymorphic locus" refers to an allele or locus where the genotype differs between individuals of a given species. Some examples of polymorphic alleles include single nucleotide polymorphisms, short tandem repeats, deletions, duplications, and inversions.

[0040] On the other hand, 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 to 8, wherein the method comprises obtaining a reference nucleic acid; isolating nucleic acid sequences comprising the 5' end and the 3' end flanking the microdeletion of interest; and replacing a central region of the reference nucleic acid sequence corresponding to the microdeletion of interest with a barcode.

[0041] As used herein, the term "nucleic acid" refers to nucleic acid in its broadest sense and is not limited to any particular form or type of nucleic acid. In some embodiments, the nucleic acid can be genomic DNA, mononucleosomal DNA, dinucleosomal DNA, trinucleosomal DNA, 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 RNAs such as miRNA, tRNA, or piwiRNA. The term nucleic acid can also include synthetic or modified nucleic acids.

[0042] In some embodiments, the reference nucleic acid is obtained from a cell line suitable for use as a positive control for detecting the one or more microdeletions.

[0043] In some embodiments, the reference nucleic acid is mononucleosomal.

[0044] In another aspect, the present disclosure relates to a method for preparing a preparation of amplified DNA derived from a sample or a fraction thereof that can be used to identify one or more microdeletions associated with a disease or condition, the method 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 a fraction thereof to obtain a spiked sample and extracting nucleic acid from the spiked sample or a fraction thereof; (c) performing targeted amplification on the spiked sample or a fraction thereof from (b) to amplify one or more target regions containing the microdeletions of interest to obtain amplicons; and (d) analyzing the amplicons or portions thereof from (c) to determine (i) whether the amplicon contains the amplified construct as a positive control, and (ii) whether the amplicon contains the one or more microdeletions of interest.

[0045] In some embodiments, constructs used as positive controls for detecting one or more microdeletions are prepared by chemical synthesis followed by PCR amplification.

[0046] 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 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.

[0047] As used herein, the term "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 comprises free DNA. In some embodiments, the biological sample is from a pregnant woman. In some embodiments, the isolated cfDNA is a mixture of fetal and maternal cfDNA. In some embodiments, the plasma sample comprises maternal and fetal free DNA, and wherein the SNP in the construct is changed to serve as a positive control for the fetal free DNA.

[0048] In some embodiments, biological samples include circulating tumor DNA (ctDNA). In some embodiments, the compositions herein are used as positive controls for detecting ctDNA and detecting or monitoring cancer or tumors. ctDNA has been found in the circulation of patients diagnosed with malignant tumors (including but not limited to lung cancer, prostate cancer, colon cancer and breast cancer). Identification of genomic instability associated with cancer that can be determined in the ctDNA of cancer patients is a potential diagnostic and prognostic tool. In one embodiment, the method of the present invention assesses microdeletions of interest in a sample, and the sample comprises a mixture of nucleic acids derived from a subject suspected or known to have cancer (e.g., cancer, sarcoma, lymphoma, leukemia, germ cell tumor and blastoma). In one embodiment, the sample is a plasma sample derived from (processed from) peripheral blood, and it comprises a mixture of cfDNA derived from normal cells and cancer cells (ctDNA). In another embodiment, the biological sample required to determine the presence or absence of a microdeletion is derived from a mixture of cancerous and non-cancerous cells from other biological fluids, including but not limited to serum, sweat, tears, sputum, urine, sputum, ear flow, lymph, saliva, cerebrospinal fluid, bone marrow suspension, vaginal flow, transcervical lavage, brain fluid, ascites, breast milk, respiratory secretions, intestinal secretions, and urogenital secretions, as well as leukapheresis samples, or tissue biopsy, swab or smear.

[0049] Chromosomal deletions involving tumor suppressor genes can play an important role in the occurrence and progression of solid tumors. The retinoblastoma tumor suppressor gene (Rb-1) is located on chromosome 13q14 and is the most widely characterized tumor suppressor gene. Alterations or loss of Rb protein expression are caused by inactivation of both gene alleles through point mutations or chromosomal deletions. Rb-i gene changes have been found 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 such tumor types often lose heterozygosity at 13q, indicating that one of the Rb-1 gene alleles is lost due to severe chromosome deletion. Chromosome 1 abnormalities, including duplications, deletions, and unbalanced translocations involving chromosome 6 and other partner chromosomes, indicate that regions of chromosome 1, particularly 1q21-1q32 and 1p11-13, may contain oncogenes or tumor suppressor genes associated with chronic and advanced myeloproliferative neoplasms pathology. Myeloproliferative neoplasms are also associated with deletions of chromosome 5. Complete loss or interstitial deletions of chromosome 5 are the most common karyotypic abnormalities in myelodysplastic syndromes (MDS). Patients with isolated del(5q) / 5q-MDS have a better prognosis than those with additional karyotypic defects, who are prone to develop myeloproliferative neoplasms (MPNs) and acute myeloid leukemia. Other candidate microdeletions associated with cancer may include the ribosomal subunit RPS14, the transcription factor Egr1 / Krox20, and the cytoskeletal remodeling protein α-catenin. Cytogenetic and allelic studies of fresh tumors and tumor cell lines have shown that allelic losses of several different regions on chromosome 3p, including 3p25, 3p21-22, 3p21.3, 3p12-13, and 3p14, are the earliest and most common genomic abnormalities in major epithelial cancers involving a wide range of 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 thought that interstitial loss or promoter hypermethylation precedes loss of 3p or the entire chromosome 3 during cancer development.

[0050] Neonates and children with Down syndrome (DS) often present with transient congenital leukemia and are at increased risk for acute myeloid leukemia and acute lymphoblastic leukemia. Chromosome 21 contains approximately 300 genes and can be associated with many structural aberrations (e.g., translocations, deletions, and amplifications) in leukemias, lymphomas, and solid tumors. In addition, genes located on chromosome 21 have been identified to play important roles in tumorigenesis. Somatic numerical aberrations and structural aberrations of chromosome 21 are associated with leukemia, and specific genes located on 21q, including RUNX1, TMPRSS2, and TFF, play a role in tumorigenesis.

[0051] In some embodiments, at least 5 microdeletions of interest are amplified in a single reaction volume, and a construct used as a positive control is prepared for each of the at least 5 microdeletions of interest. In some embodiments, disclosed herein is a method for preparing a preparation of amplified DNA derived from a sample or fraction thereof that can be used to identify one or more microdeletions associated with a disease or condition, and the one or more microdeletions include 22q11.2 deletion (DiGeorge syndrome), chromosome 5p15.2 (Cry-a-cat syndrome), 1p36 deletion, 15q11.2-q13 deletion (Prader-Willi syndrome) and / or 15q11-q13 (Angelman syndrome).

[0052] 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-Giedion / trichodactyly type II, 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-Magilli syndrome type 2, Magenis), MillerDieker syndrome, RCAD (17q12 deletion), 17q21.31 microdeletion, 18q21.2 microdeletion: Pitt-Hopkins syndrome, DiGeorge, 22q11.21 microdeletion, 22q11.2 microdeletion, Phelan McDermid 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-Shaffer syndrome, 13q14.2 deletion, retinoblastoma with ID, 13q32 deletion-HPE5, PKD1 / TSC2 continuous deletion syndrome, 17p13.3 distal microdeletion, 17p13.3 distal microdeletion, 17q21.31 microdeletion, isochromosome, 21q22.3 microdeletion: holoprosencephaly 1, PelizaeusMerzbacher XL.

[0053] In some embodiments, the microdeletion of interest may be associated with cancer.

[0054] In some embodiments, disclosed herein is a method for preparing a preparation of amplified DNA derived from a sample or a fraction thereof that can be used to identify one or more microdeletions associated with a disease or condition, the method 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.

[0055] In some embodiments, disclosed herein is a method for preparing a preparation of amplified DNA derived from a sample or a fraction thereof that can be used to identify one or more microdeletions associated with a disease or condition, by determining the efficiency and error rate of each amplification reaction using the positive control, wherein the efficiency and the error rate are used to determine the presence of the one or more microdeletions of interest.

[0056] Microdeletions of interest can be identified by combining the positive control constructs disclosed herein with the methods for identifying microdeletions described in US 17 / 252205, which is incorporated herein by reference in its entirety.

[0057] In some embodiments, the present disclosure provides the amount of the construct to be added to the sample, which is determined by: (a) mixing mononucleosomal DNA from a normal female cell line with the construct in a certain ratio range to generate a titration series to determine the detection limit; (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 and mononucleosomal DNA from a normal cell line that allows detection of the construct.

[0058] Some embodiments of the invention are kits comprising a composition comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, or 50 different engineered nucleic acid constructs at the low end and 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 50, 100, or 250 different engineered nucleic acid constructs at the high end, wherein 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 microdeletion of interest corresponding to a 22q11.2 deletion; (ii) a second engineered nucleic acid construct comprising a barcode replacing a microdeletion of interest corresponding to a 5p15.2 deletion; (iii) a third engineered nucleic acid construct comprising a barcode replacing a microdeletion of interest corresponding to a 1p36 deletion; (iv) a fourth engineered nucleic acid construct comprising a barcode replacing a microdeletion of interest corresponding to a 15q11.2-q13 deletion; and / or (v) a fifth engineered nucleic acid construct comprising a barcode replacing a microdeletion of interest corresponding to a 15q11-q13 deletion. The kit may include primers for amplifying one or more microdeletions of interest and a corresponding engineered nucleic acid construct used as a positive control.

[0059] Isolation of nucleic acids

[0060] As used herein, the term "isolation" refers to the physical separation of target genetic material from other biological material. It can also refer to partial separation, in which the isolated target is separated from some or most but not all of the biological material.

[0061] Isolation of mononucleosomal DNA: It has been shown that cfDNA can exist in the form of nucleosome complexes, in which DNA is tightly wrapped around histones. The mononucleosome complex consists of about 130 to about 170bp of DNA wrapped around a single nucleosome. The term "mononucleosome" refers to a fragment of chromosomal DNA containing a single nucleosome or refers to a nucleic acid sequence of the size of a mononucleosomal DNA, which is about 130 to about 170bp in size. The term "sub-mononucleosome" refers to a fragment of chromosomal DNA with a molecular size of less than about 130bp, which is expected to be derived from a complete nucleosome. cfDNA can also be integrated and present in lipid vesicles (such as exosomes).

[0062] Chromosomal DNA consists of DNA wrapped around histone protein complexes that form nucleosomes. Nucleosomes protect the DNA, so fragmented chromosomal DNA is often found as multiples of nucleosomes.

[0063] Many methods known to those of ordinary skill in the art can be used to separate free DNA from biological samples. Such methods include, but are not limited to, organic liquid phase extraction, which utilizes phenol and phenol-chloroform mixtures to decompose nucleoprotein complexes and isolate proteins and lipids into an organic phase, while highly hydrophilic DNA and RNA are distributed into an aqueous phase in a very pure form. Other methods include the use of agarose hydrogels, such as those described in EM Southern (J. Mol. Biol. (1975) 94: 51-70) and Vogelstein and Gillespie (PNAS, USA (1979) 76: 615-619), which are incorporated herein in their entirety. Another method is to capture DNA on a solid phase material, as described in Boom et al. (J Clin Micro. (1990) 28 (3): 495-503), which are incorporated herein in their entirety. General methods for DNA isolation can be found in Sambrook J, Russel DW (2001). Molecular Cloning: A Laboratory Manual 3rd Edition Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY, which is incorporated herein.

[0064] In one specific aspect, cfDNA is extracted by using an ion exchange based bead method.

[0065] Further methods, described in detail below, can be used to enrich for DNA fragments within a specific molecular size range.

[0066] Size selection / exclusion methods

[0067] The present disclosure relates to methods involving size selection by gel electrophoresis, paramagnetic beads, spin columns, salt precipitation, or biased amplification.

[0068] In some embodiments, the size exclusion step of the method disclosed herein is performed by using gel electrophoresis to separate cfDNA samples according to size and selecting a determined size range. Gel electrophoresis is a method recognized in the art to separate DNA molecules according to the size of DNA molecules by applying an electric field to a gel (such as an agarose gel) (at this time, the DNA molecules will move through the gel to the positively charged anode). The size of the DNA molecule will determine the speed at which the DNA molecule migrates through the gel. A standard mixture of DNA molecules with a predetermined size can be applied to the gel to identify the size of the DNA. Then, the DNA molecules of the desired size can be extracted and purified using well-known techniques (such as those disclosed in Sambrook J, Russel DW (2001) incorporated into Molecular Cloning: A Laboratory Manual 3rd Edition Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY). In some embodiments, size selection is performed on an automated high-throughput gel electrophoresis system (such as a Pippin or Costal Genomics system).

[0069] In some embodiments, the size exclusion step of the method disclosed herein is performed by using paramagnetic beads. The size selection of DNA fragments using paramagnetic beads 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), which is incorporated herein. In short, the method is based on the total charge of each molecule affected by the size of the DNA fragments, and larger DNA has a larger charge, which promotes their electrostatic interaction with the beads and replaces smaller DNA fragments. Therefore, by controlling the composition of the buffer solution used to mix the beads and DNA, the beads can be bound to DNA within a specific size range. The most famous and most widely used method is solid phase reversible separation (SPRI) selection, which uses carboxyl-coated paramagnetic beads in the presence of high salt and crowding agent polyethylene glycol (PEG) to promote controlled adsorption, and is configured to bind to DNA within a certain molecular weight range by changing the PEG concentration. By subjecting the source DNA to various binding and elution protocols in the presence of different amounts of PEG, DNA molecules of different lengths can be distributed. TM The beads were used for the size exclusion step.

[0070] In certain embodiments, the size exclusion step of the method disclosed herein is performed by using a centrifugal column. The centrifugal column comprises a material that absorbs molecules according to molecular size. The centrifugal column material comprises a hole of a certain size, and molecules of a size higher than the cut-off size determined by the pore size will not enter the hole and will be eluted with the void volume of the column. Different types of column materials can be selected to achieve absorption or exclusion of DNA molecules within different size ranges. In certain embodiments, the centrifugal column material comprises siliceous material, silica gel, glass, glass fiber, zeolite, aluminum oxide, titanium dioxide, zirconium dioxide, kaolin, gelatinous silica, magnetic particles, ceramics, polymer support materials or combinations thereof. In certain embodiments, the centrifugal column material comprises glass fiber.

[0071] In some embodiments, the spin column can be used for size exclusion by using different binding buffers configured to provide low or high stringency binding conditions when the DNA sample is applied to the spin column, as described in PCT Patent Application No. PCT / US2019 / 18274 filed on 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 the binding of low molecular weight DNA fragments, while high stringency binding conditions will configure the spin column to promote the binding of low molecular weight DNA fragments.

[0072] In some embodiments, low and / or high stringency binding buffer comprises a nitrile compound selected from acetonitrile (ACN), propionitrile (PCN), butyronitrile (BCN), isobutyronitrile (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).

[0073] In some embodiments, the low and / or high stringency binding buffer comprises a chaotropic compound selected from GnCl, urea, thiourea, guanidine thiocyanate, NaI, guanidine isothiocyanate, D- / L-arginine, Li+, Na+, K+, perchlorate, or a combination thereof. The low and / or high stringency binding buffer may comprise, for example, a chaotropic compound (e.g., GnCl) at about 5 M to about 8 M, or about 5.6 M to about 7.2 M, or about 6 M.

[0074] Binding buffer can also include alcohol, chelating agent and detergent. In certain embodiments, alcohol is propanol. In certain embodiments, chelating compound includes ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis (2-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA), citric acid, N, N, N', N'-tetra (2-pyridylmethyl) ethylenediamine (TPEN), 2,2'-bipyridine, deferoxamine methanesulfonate (DFOM), 2,3-dihydroxysuccinic acid (tartaric acid) or its combination. In certain embodiments, detergent can be Triton X-100, Tween 20, N-lauroylsarcosine, sodium dodecyl sulfate (SDS), dodecyl dimethyl phosphine oxide, sorbitol monopalmitate, decyl hexaglycol, 4-nonylphenyl-polyethylene glycol or its combination. In certain embodiments, detergent is Triton X-100.

[0075] In some embodiments, the size exclusion step of the method disclosed herein is performed by using salt precipitation. Larger DNA molecules will precipitate at lower salt concentrations than smaller DNA molecules. By changing the concentration of salt in the precipitation buffer, DNA molecules of different size ranges can be separated.

[0076] Amplification method

[0077] In some embodiments, the method includes performing targeted amplification to identify microdeletions of interest. In some embodiments, the targeted amplification for identifying microdeletions of interest includes performing amplification of multiple amplification reactions. In some embodiments, the amplification reaction is a single reaction performed in parallel. In some embodiments, the target locus is amplified by performing multiple amplifications of multiple target loci in a single reaction. In some embodiments, the amplification reaction for identifying microdeletions of interest is performed by using multiple amplifications of multiple target loci in a single reaction. In some embodiments, at least 5 microdeletions of interest are identified by performing multiple amplifications in a single reaction volume.

[0078] In some embodiments, the reaction mixture used to perform multiplex amplification includes an engineered construct that serves as a positive control for identifying a microdeletion of interest.

[0079] Multiplex amplification reactions can be set up as single reactions, or can be set up as pools of different subset multiplex reactions. The multiplex reaction methods provided herein, such as the large-scale multiplex PCR disclosed herein, provide exemplary processes for performing amplification reactions to help achieve improved multiplexing and thus sensitivity levels.

[0080] In certain illustrative embodiments, nucleic acid sequence data is produced by high-throughput DNA sequencing of multiple copies of a series of amplicons produced using a multiplex amplification reaction, wherein each amplicon of the amplicon series spans at least one polymorphic locus in a set of polymorphic loci, and wherein each of the polymorphic loci of the set is amplified. For example, in these embodiments, multiplex PCR can be performed to amplify amplicons across 5 microdeletion regions, and wherein the amplicons comprise multiple polymorphic loci.

[0081] In some embodiments, amplification is performed using direct multiplex PCR, sequential PCR, nested PCR, double nested PCR, one-sided and half-sided nested PCR, fully nested PCR, one-sided fully nested PCR, one-sided nested PCR, semi-nested PCR, semi-nested PCR, triple semi-nested PCR, semi-nested PCR, single-sided semi-nested PCR, reverse semi-nested PCR method or single-sided PCR, which are described in U.S. application No. 13 / 683,604 filed on November 21, 2012, U.S. publication No. 2013 / 0123120, U.S. application No. 13 / 300,235 filed on November 18, 2011, U.S. publication No. 2012 / 0270212 and U.S. serial No. 61 / 994,791 filed on May 16, 2014, which are hereby incorporated by reference in their entirety.

[0082] In one embodiment, the multiplex PCR assay is designed to amplify the microdeletion region of interest and / or to amplify SNPs within or flanking the microdeletion of interest.

[0083] In some embodiments, a method of amplifying a target locus in a nucleic acid sample involves (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 produce a single reaction mixture; and (ii) subjecting the reaction mixture to primer extension reaction conditions (such as PCR conditions) to produce an amplification product comprising the target amplicon. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% of the target locus is 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 product is a primer dimer. In some embodiments, the primer is in solution (such as dissolved in a liquid phase rather than a solid phase). In some embodiments, the primer is in solution and is not fixed on a solid phase carrier. In some embodiments, the primer is not a part of a microarray.

[0084] In certain embodiments, at least 1 / 2 of the multiple amplification reactions are performed under limiting primer conditions. In certain embodiments, limiting primer concentrations are used for 1 / 10, 1 / 5, 1 / 4, 1 / 3, 1 / 2 or all of the multiple reactions. Factors to consider when implementing limiting primer conditions in an amplification reaction (such as PCR) are provided herein.

[0085] PCR reaction conditions can be, for example, STAR (specific target amplification reaction) scheme disclosed in US17 / 545881, which is incorporated herein in its entirety. For example, for at least 10%, 20%, 25%, 30%, 40%, 50%, 06%, 70%, 75%, 80%, 90%, 95% or 100% of the primers in the set of primers, the PCR reaction can be carried out at an annealing temperature between 1°, 2°, 3°, 4°, 5°, 6°, 7°, 8°, 9° or 10° (at the lower end of the range) and 2°, 3°, 4°, 5°, 6°, 7°, 8°, 9°, 10°, 11°, 12°, 13°, 14° or 15° (at the upper end of the range) higher than the melting temperature.

[0086] In certain embodiments, where the amplification reaction is a PCR reaction, the length of the annealing step in the PCR reaction is between 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 upper end of the range. In certain embodiments, the primer concentration in the amplification (such as a PCR reaction) is between 1 nM and 10 nM. In addition, in an exemplary embodiment, the primers in the set of primers are designed to minimize primer dimer formation.

[0087] Therefore, in the examples of any method comprising an amplification step herein, the amplification reaction is a PCR reaction, the annealing temperature is 1°C to 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 minutes to 60 minutes, the primer concentration in the amplification reaction is 1 nM to 10 nM, and the primers in the primer set are designed to minimize primer dimer formation. In another aspect of this example, multiple amplification reactions are performed under limiting primer conditions.

[0088] In some embodiments, a range of multiplex reactions are performed: between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 500, 1000, 2500, 5000, 10,000, 20,000, 25000, 50000 at the lower end of the range and between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 500, 1000, 2500, 5000, 10,000, 20,000, 25000, 50000 and 100,000 at the upper end of the range.

[0089] In one embodiment, multiplex PCR assays are designed to amplify SNPs within or flanking microdeletions of interest, and these assays are used in a single reaction to amplify DNA. The number of PCR assays can be 1 and 10, 5 and 10, 10 and 50, 50 and 200 PCR assays, 5 and 20 PCR assays, 100 and 1,000 PCR assays, 1,000 and 10,000, or 1,000 and 20,000 or more than 20,000 PCR assays (5 to 20-fold, 100 to 1,000-fold, 1,00 to 5,000-fold, 1,000 to 10,000-fold, 1,000 to 20,000-fold or more than 20,000-fold, 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: a plasma sample, a chorionic villus sample, an amniocentesis sample, a single or small number of cells, other body fluids or tissues, cancer or other genetic material.

[0090] The SNP frequency of each locus can be determined by sequencing the amplicon by cloning or some other method. All measured allele frequency distributions or statistical analysis of the ratio can be used to determine whether the sample contains one or more trisomy of the chromosomes included in the test. In another embodiment, the original cfDNA sample is divided into two samples and 5,000 parallel determinations are performed. In another embodiment, the original cfDNA sample is divided into n samples and parallel (about 10,000 / n) determinations are performed, wherein n is between 2 and 12, or between 12 and 24, or between 24 and 48, or between 48 and 96.

[0091] The genetic data obtained from the multiplex PCR were analyzed using bioinformatics methods. 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.

[0092] Hybridization capture method

[0093] In some embodiments, the method includes performing hybridization capture to select for a plurality of polymorphic loci on the selectively enriched DNA prior to determining the sequence of the selectively enriched DNA.

[0094] In some embodiments, preferentially enriching DNA at multiple polymorphic loci comprises: obtaining multiple hybridization capture probes targeting the polymorphic loci; hybridizing the hybridization capture probes to DNA in the sample; and physically removing some or all of the unhybridized DNA from the first DNA sample.

[0095] In certain embodiments, the hybridization capture probe is designed to hybridize with the region that is located at the flank of the polymorphic locus but does not overlap with it. In certain embodiments, the hybridization capture probe is designed to hybridize with the region that is located at the flank of the polymorphic locus but does not overlap with it, and wherein the length of the flank capture probe can be selected from the group consisting of the following items: 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 certain embodiments, the hybridization capture probe is designed to hybridize with the region that overlaps with the polymorphic locus, and wherein a plurality of hybridization capture probes include at least two hybridization capture probes for each polymorphic locus, and wherein each hybridization capture probe is designed to complement the different alleles at the polymorphic locus.

[0096] Nucleic acid sequencing

[0097] Sample nucleic acid can be further analyzed by genotyping microarray and high-throughput sequencing. Some high-throughput sequencing methods include Sanger DNA sequencing, pyrophosphate sequencing, ILLUMINA SOLEXA platform, ILLUMINA's GENOMEANALYZER or APPLIED BIOSYSTEM's 454 sequencing platform, HELICOS's TRUE SINGLE MOLECULESEQUENCING platform, HALCYON MOLECULAR's electron microscope sequencing method, or any other sequencing method. In certain embodiments, high-throughput sequencing is carried out on Illumina NextSeq. In certain embodiments, high-throughput sequencing methods include NovaSeq, BGI / MGI sequencing system, Omniome / PacBio sequencing system, Oxford Nanopore sequencing system or IonTorrent sequencing system.

[0098] In some embodiments, the sequence of the selectively enriched DNA is determined by performing a microarray analysis. In one embodiment, the microarray can be an ILLUMINA SNP microarray or an AFFYMETRIX SNP microarray.

[0099] In some embodiments, the sequence is determined by performing quantitative PCR (qPCR) or digital droplet PCR (ddPCR) analysis. qPCR measures the fluorescence intensity at a specific time (generally after each amplification cycle) to determine the relative number of target molecules (DNA). ddPCR measures the actual number of molecules (target DNA) because each molecule is in a droplet, making it a discrete "digital" measurement. It provides absolute quantification because ddPCR measures the positive fraction of the sample, that is, the number of droplets that fluoresce due to correct amplification. This positive fraction accurately indicates the initial amount of template nucleic acid.

[0100] As used herein, the term "adapter" or "connection adapter" or "library tag" is a DNA molecule containing a universal primer sequence that can be covalently linked to the 5-primary end and 3-primary end of a target double-stranded DNA molecule population. In some embodiments, the addition of the adapter provides a universal primer sequence for the 5-primary end and 3-primary end of the target population, from which PCR amplification can be performed, using a single pair of amplification primers to amplify all molecules of the target population. Disclosed herein is a method that allows targeted amplification of more than one hundred to tens of thousands of target sequences (e.g., SNP loci) from genomic DNA obtained from plasma. The amplified sample can be relatively free of primer dimer products and have low allele bias at the target locus. If during or after amplification, the product is attached to a sequencing-compatible adapter, analysis of these products can be performed by sequencing. These methods are more fully described in U.S. Patent Publications 20170242960 and 20180025109 and U.S. Patent 9,163,282, which are incorporated herein.

[0101] In certain embodiments, the adapter or primer described herein may include one or more molecular barcodes. Molecular barcodes or molecular index sequences can be used for next generation sequencing to reduce the quantitative bias introduced by replication. In next generation sequencing, each nucleic acid fragment can be marked with a molecular barcode or a molecular index sequence. Sequence reads with different molecular barcodes or molecular index sequences represent different original nucleic acid molecules. By referring to molecular barcodes or molecular index sequences, PCR artifacts (such as sequence changes produced by polymerase errors) that are not present in the original nucleic acid molecules can be identified and separated from the true variants / mutations present in the original nucleic acid molecules.

[0102] In some embodiments, the molecular barcode is introduced by ligating an adaptor carrying the molecular barcode to the isolated cfDNA to obtain adaptor-ligated and molecularly barcoded DNA. In some embodiments, the molecular barcode is introduced by amplifying the adaptor-ligated DNA with primers carrying the molecular barcode to obtain amplified adaptor-ligated and molecularly barcoded DNA.

[0103] In some embodiments, a molecular barcode adapter or primer may comprise a universal sequence followed by a molecular barcode region, optionally followed by a target-specific sequence in the case of a primer. The sequence 5' of the molecular barcode can be used for subsequent PCR amplification or sequencing, and may comprise a sequence that can be used to convert the amplicon into a library for sequencing. Random molecular barcode sequences can be generated in a variety of ways. A preferred method synthesizes molecular tag adapters or primers in a manner that incorporates all four bases into the reaction during synthesis of the barcode region. IUPAC DNA ambiguity codes can be used to specify combinations of all or various bases. In this way, a collection of synthesized molecules will contain a random mixture of sequences in the molecular barcode region. The length of the barcode region will determine how many adapters or primers will contain unique barcodes. The number of unique sequences is related to the length of the barcode region and is N. L , where N is the number of bases, typically 4, and L is the length of the barcode. A five-base barcode can produce up to 1024 unique sequences; an eight-base barcode can produce 65,536 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 that sequence a single molecule directly, or methods that amplify a single molecule to form a clone that can be detected by a sequencing instrument, but still represents a single molecule, referred to herein as clonal sequencing.

[0104] 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 to identify a single sample nucleic acid molecule from a population of nucleic acid molecules (i.e., a member of a population) after sample processing for a sequencing reaction. MITs are described in detail in U.S. Pat. No. 10,011,870 to Zimmermann et al., which is incorporated herein by reference in its entirety. Unlike prior art methods that involve unique identifiers and teach having multiple unique identifiers greater than the number of sample nucleic acid molecules in a sample so that each sample nucleic acid molecule is labeled with a unique identifier, the present disclosure generally involves many more sample nucleic acid molecules than multiple MITs in a set of MITs. In fact, the methods and compositions herein may include more than 1,000, 1x10 MITs for each different MIT in a set of MITs. 6 , 1x10 9 Or even more starting molecules. However, these methods can still identify a single sample nucleic acid molecule that produces a labeled nucleic acid molecule after amplification.

[0105] General Definition

[0106] As used in the description of the 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 indicates otherwise.

[0107] As used herein, the term "about" when referring to a measurable value such as an amount or concentration, etc., is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5% or even 0.1% of the specified amount.

[0108] The terms or "acceptable," "effective," or "sufficient" when used to describe the selection of any component, range, dosage form, etc. disclosed herein, mean that the component, range, dosage form, etc. is suitable for the disclosed purpose.

[0109] Also as used herein, "and / or" refers to and encompasses all possible combinations of any one and one or more of the associated listed items, and when interpreted in alternatives ("or") refers to and encompasses the lack of a combination.

[0110] As used herein, the term "comprising" is intended to mean that the composition and method include the described elements, but do not exclude other elements. As used herein, the transition phrase "consisting essentially of..." (and grammatical variations) should be interpreted as covering the materials or steps "and those that do not substantially affect the basic and novel features of the embodiments". See In re Herz, 537 F.2d 549, 551-52, 190 USPQ461, 463 (CCPA 1976) (emphasis in the original); see also MPEP §2111.03. Therefore, the term "consisting essentially of..." as used herein should not be interpreted as being equivalent to "comprising". "Consisting of..." should mean excluding other ingredients more than trace elements and a large number of method steps for applying the compositions disclosed herein. The aspects defined by each of these transition terms are within the scope of the present disclosure.

[0111] Working Example

[0112] Example 1

[0113] This example illustrates the construction of an engineered construct for use as a positive control in a method for identifying a microdeletion of interest, and the use of the construct to identify a microdeletion of interest.

[0114] The overall method used to build the engineered constructs is shown in Figure 1 This construct is used as a positive control in a method for identifying a microdeletion of interest. First, a reference DNA sequence containing the microdeletion region of interest is obtained. Figure 1In the figure, the "^" symbol represents a SNP, and the "v" represents an inversion SNP. Secondly, the region within the microdeletion region is deleted and replaced with a barcode of 6 to 9 base pairs. The 5' and 3' flanking regions of the central microdeletion region contain binding sites for primers suitable for use with, for example, STAR (specific target amplification reaction) primers targeting microdeletions. Primers can be designed to identify 22q11.2 deletions (DiGeorge syndrome), chromosome 5p15.2 (Call of Cats syndrome), 1p36 deletions, 15q11.2~q13 deletions (Prader-Willi syndrome) and / or 15q11~q13 (Angelman syndrome).

[0115] Constructs were prepared by using chemical synthesis and subsequent PCR amplification of the synthetic constructs.

[0116] The engineered construct used as a positive control is mixed with a nucleic acid sample from a subject to obtain a spiked sample. The amount of the construct to be added to the sample is determined by: (a) mixing mononucleosomal DNA from a normal female cell line with the construct in a certain ratio range to generate a titration series to determine the detection limit; (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 a normal cell line that allows detection of the construct. The identified amount of the construct is added to a sample from a subject, and targeted amplification of the microdeletion of interest is performed. The construct will be amplified as a positive control and can be identified by replacing the barcode of the microdeletion region. The microdeletion of interest can be amplified in a single reaction volume, in which case a composition containing all the positive control constructs of the microdeletion of interest is added to the sample. The amplification of the positive control construct can determine the efficiency and error rate, which can be used to calibrate the amplification results of the sample nucleic acid to improve the accuracy of identifying the microdeletion of interest.

[0117] Demonstration that the SNP assay flanking the barcode replaces the microdeletion in the engineered construct works in the STAR reaction, indicating that the assays flanking and within the deletion are functional. Therefore, this construct can be used as a positive control.

Claims

1. A composition comprising an engineered nucleic acid construct, the construct being used 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, wherein the construct comprises a 5' terminal region, a central region, and a 3' terminal region, wherein the 5' terminal region and the 3' terminal region comprise reference sequences flanking the microdeletion of interest, wherein the central region of the construct is a DNA barcode, and wherein the barcode replaces the microdeletion sequence of the reference nucleic acid sequence.

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

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

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

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

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

8. The composition of claim 3, wherein the reference nucleic acid sequence is maternal DNA, and wherein the SNP of interest is altered to allow the construct to serve as a positive control for progeny DNA.

9. 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. 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, wherein the method comprises obtaining a reference nucleic acid; isolating nucleic acid sequences comprising the 5' end and the 3' end flanking the microdeletion of interest; and replacing a central region of the reference nucleic acid sequence corresponding to the microdeletion of interest with a barcode.

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

12. The method of claim 10, wherein the sample is a plasma sample and contains free DNA.

13. The method of claim 10, wherein the sample comprises circulating tumor DNA (ctDNA). The method of claim 10 , wherein the sample comprises cells and / or tissue.

15. 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 mononucleosomal.

18. A method for preparing a preparation of amplified DNA derived from a sample or fraction thereof that can be used to identify one or more microdeletions associated with a disease or condition, the method 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 fraction thereof to obtain a spiked sample and extracting nucleic acid from the spiked sample or a fraction thereof; (c) performing targeted amplification on the spiked sample or a fraction thereof from (b) 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 amplification construct as a positive control, and (ii) whether the amplicon contains the one or more microdeletions of interest.

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

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

21. The method of claim 20, wherein at least 5 microdeletions of interest are amplified in a single reaction volume, and wherein a construct for use as a positive control is prepared for each of the at least 5 microdeletions of interest.

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

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

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

26. The method of claim 18, wherein the amount of the construct to be added to the sample is determined by: (a) mixing DNA from a normal female cell line with the construct 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 a normal cell line that allows detection of the construct.

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

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

29. A method of preparing a sample comprising nucleic acid, the method comprising spiking the sample with a composition according to claims 1 to 9.

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

31. The method of any one of claims 18 to 30, wherein detection of the SNPs flanking the barcode that replace the microdeletion in the engineered construct in the amplicon indicates that the assay for detecting SNPs flanking the microdeletion and within the microdeletion is functional, thereby confirming that the engineered construct can be used as the positive control.

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