Method for non-invasive prenatal screening using cell-free DNA extraction

Abstract

This invention relates to a method for non-invasive prenatal screening using cell-free DNA extraction. Specifically, the invention provides a method and system for extracting cell-free DNA from liquid biological samples. The method can be used for determining fetal DNA fractions and for non-invasive prenatal screening of fetal aneuploidy and analysis of other types of cell-free DNA.

Description

[0001] This application is a divisional application of Chinese invention patent application filed on October 25, 2016, with Chinese application number 201680076102.9 and invention title "Method for Non-invasive Prenatal Screening by Cell-Free DNA Extraction". Technical Field

[0002] This invention provides a method and system for cell-free DNA extraction from liquid biological samples. The method can be used for determining fetal DNA fractions and for non-invasive prenatal screening of fetal aneuploidy and analysis of other types of cell-free DNA. Background Technology

[0003] Autosomal trisomy occurs in approximately 1 in 500 live deliveries. Most of these trisomy occurs at chromosomes 13, 18, and 21. Sex chromosome aneuploidy also occurs in approximately 1 in 500 live deliveries. Current maternal serum screening markers detect only 85-95% of chromosomal abnormalities and have a false positive rate as high as 5 or 6%. Amniocentesis and CVS are highly accurate methods for identifying chromosomal abnormalities, but they carry procedural risks associated with miscarriage. These risks often outweigh the benefits of the procedure, making them unsuitable as routine screening tools. The aim of non-invasive prenatal testing is to provide accurate screening tools for detecting fetal genetic defects without the risks associated with traditional methods of fetal genetic testing.

[0004] Detection and quantification of fetal nucleic acids in maternal plasma is a non-invasive alternative method for prenatal genetic diagnosis. Effective diagnosis depends on the quantity and quality of the isolated DNA, which affects the sensitivity and reproducibility of the assay. Summary of the Invention

[0005] This article provides methods for improving the quantity, quality, and consistency of cell-free DNA (cfDNA) isolates from liquid biological samples. In a particular embodiment, the liquid biological sample is obtained from a pregnant mammal (e.g., a human patient) for noninvasive prenatal diagnosis of fetal aneuploidy.

[0006] In some embodiments, this method can be used for cancer diagnosis. In some embodiments, liquid biological samples are obtained from patients diagnosed with or suspected of having cancer, and are used to isolate and detect circulating tumor DNA. In some embodiments, the test can be applied to the general population as a screening test to predict the risk of having cancer.

[0007] In some embodiments, this document provides a method for improving the yield of cell-free DNA (cfDNA) isolated from liquid biological samples from mammals, comprising separating cfDNA from the sample using magnetic beads that reversibly bind cfDNA, and at least two types of magnetic separation steps for manipulating the magnetic beads. For example, in some embodiments, cfDNA binds to magnetic beads, and the cfDNA-bound magnetic beads are sequentially transferred from a sample tube to one or more containers, such as containers containing a washing solution or a container containing an elution solution. The eluted cfDNA is separated from the magnetic beads by contacting the outer surface of the container containing the eluted cfDNA and the elution solution containing the magnetic beads with a magnetic plate and collecting the elution solution containing the eluted magnetic beads. In some embodiments, the magnetic plate comprises a ring magnet that causes the magnetic beads to adhere to the container wall, thereby allowing the collection of the elution solution containing the eluted cfDNA.

[0008] In some embodiments, this document provides a method for isolating cell-free DNA from liquid biological samples from mammals, comprising the following steps:

[0009] a) Contact a liquid biological sample containing cfDNA with multiple reversibly DNA-binding magnetic beads.

[0010] b) Separate the cfDNA-bound magnetic beads from the unbound components of the liquid biological sample;

[0011] c) Transfer the magnetic beads bound to the cfDNA to a container containing an elution solution;

[0012] d) The magnetic beads are separated from the elution solution by contacting a container containing the cfDNA and the magnetic beads in the elution solution with a magnetic plate and collecting the elution solution containing the cfDNA.

[0013] In some embodiments, the cfDNA-bound magnetic beads are separated from the unbound components of the liquid biological sample by transferring the cfDNA-bound magnetic beads from the liquid biological sample to another container. In some embodiments, the cfDNA-bound magnetic beads are transferred directly from the liquid biological sample to a container containing the elution solution. In some embodiments, the cfDNA-bound magnetic beads transferred from the liquid biological sample are transferred to a container containing a washing solution.

[0014] In some embodiments, after step (b) and before step (c), the cfDNA-bound magnetic beads are transferred to a washing solution. In some embodiments, the cfDNA-bound magnetic beads are magnetically transferred to a washing solution. In some embodiments, the cfDNA-bound magnetic beads are sequentially transferred to two or more washing solutions. In some embodiments, the cfDNA-bound magnetic beads are sequentially and magnetically transferred to two or more washing solutions. In some embodiments, the two or more washing solutions are identical. In some embodiments, the two or more washing solutions comprise two or more different washing solutions.

[0015] In some embodiments, the cfDNA-bound magnetic beads are separated from the unbound components of the liquid biological sample by inserting a rod-shaped magnet into the sample to magnetically bind the cfDNA-bound magnetic beads, and the magnetically bound beads are deposited in another container. In some embodiments, the container contains a washing solution. In some embodiments, the container contains an elution solution.

[0016] In some embodiments, the cfDNA-bound magnetic beads are transferred to a washing solution by inserting a rod-shaped magnet into the liquid biological sample to magnetically bind the cfDNA-bound magnetic beads, and the magnetically bound beads are deposited in a container containing the washing solution.

[0017] In some embodiments, the cfDNA-bound magnetic beads are transferred to an elution solution by inserting a rod-shaped magnet into an ethanol wash solution containing cfDNA-bound magnetic beads, and the magnetically bound magnetic beads are deposited in a container containing the elution solution. In some embodiments, the washed cfDNA-bound magnetic beads are incubated in the elution solution for a sufficient time to elute the cfDNA from the magnetic beads. A suitable elution solution releases the bound cfDNA from the magnetic beads. Exemplary elution solutions include water or buffer solutions such as Tris buffer (e.g., 10 mM Tris, pH 7.0-8.0) or TRIS-EDTA buffer.

[0018] In some implementations, the rod-shaped magnet is surrounded by a tip cover, so that the magnet does not come into direct contact with the liquid biological sample, washing solution, or elution solution.

[0019] In some implementations, the transfer of magnetic beads from one container to another is automated.

[0020] In some embodiments, the surface of the magnetic beads contains one or more functional groups that reversibly bind DNA. In some embodiments, the magnetic beads comprise a magnetic metal oxide and a polymer. In some embodiments, the magnetic metal oxide is an oxide of iron, cobalt, or nickel, or a combination thereof. In some embodiments, the polymer is cross-linked polystyrene. In some embodiments, the surface of the magnetic beads is coated with a silicone coating. In some embodiments, the magnetic beads comprise a polymer core, a magnetic layer comprising a magnetic metal oxide coating the polymer, and a silicone outer layer covering the magnetic layer.

[0021] In some embodiments, the magnetic beads are of uniform diameter. In some embodiments, the magnetic beads are of non-uniform diameter. In some embodiments, the diameter of the magnetic beads is approximately 1 µm.

[0022] In some implementations, magnetic beads are incubated for a sufficient time to bind cfDNA in liquid biological samples to generate cfDNA-bound magnetic beads.

[0023] In some implementations, the magnetic plate contains a ring magnet.

[0024] In some embodiments, the liquid biological sample is a plasma sample. In some embodiments, the liquid biological sample is prepared by removing cells from a blood sample.

[0025] In some implementations, the liquid biological sample is obtained from humans.

[0026] In some implementations, the liquid biological sample is obtained from a pregnant female.

[0027] In some implementations, the volume of the liquid biological sample is 0.5-2 mL.

[0028] In some embodiments, the volume of the elution solution is less than 200 µl. In some embodiments, the volume of the elution solution is less than or about 150 µl, less than or about 100 µl, less than or about 90 µl, less than or about 80 µl, less than or about 70 µl, less than or about 60 µl, less than or about 50 µl, or less than or about 40 µl.

[0029] In some embodiments, step (a) is performed in a porous plate. In some embodiments, the washing and elution steps are performed in a porous plate of the same size. In some embodiments, the porous plate is a 24-well deep-hole plate.

[0030] In some embodiments, at least two replicate liquid biological samples containing cfDNA are contacted with a plurality of magnetic beads. In some embodiments, the magnetic beads containing the cfDNA in each replicate sample are aggregated after step (a).

[0031] In some embodiments, at least one of the two or more washing solutions is an ethanol washing solution. In some embodiments, cfDNA-bound magnetic beads are transferred to the ethanol washing solution by inserting a rod-shaped magnet into the washing solution to magnetically bind cfDNA-bound magnetic beads, and the magnetically bound beads are deposited in a container containing the ethanol washing solution. In some embodiments, the method further includes transferring the washed cfDNA-bound magnetic beads to a container containing a second ethanol washing solution.

[0032] In some embodiments, the washed cfDNA-bound magnetic beads are incubated in elution solution for about 1 to about 10 minutes. In some embodiments, the cfDNA-bound magnetic beads are incubated in elution solution for at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, or at least 10 minutes. In some embodiments, heat is applied to the magnetic beads to elute the cfDNA. Detailed Implementation

[0033] certain terms

[0034] To facilitate understanding of this disclosure, some terms and phrases are defined below.

[0035] Unless otherwise stated, the singular forms “a,” “an,” and “the” used herein also include the plural forms. Thus, for example, reference to “oligonucleotide” includes multiple oligonucleotide molecules, reference to “tag” refers to one or more tags, reference to “probe” refers to one or more probes, and reference to “nucleic acid” refers to one or more polynucleotides.

[0036] Unless otherwise stated, when referring to numerical values, the term “about” as used herein means adding or subtracting 10% of the listed values.

[0037] As used herein, a “vector” or “genetic vector” is an individual that possesses at least one copy of an allele of a genetic determinant that is involved in the expression of a particular genotype.

[0038] As used herein, the terms “amplification” or “amplify” include methods for copying target nucleic acids, thereby increasing the copy number of a selected nucleic acid sequence. Amplification can be exponential or linear. The target nucleic acid can be DNA or RNA. The sequence amplified in this manner forms an “amplification product,” also known as an “amplifier.” While the exemplary methods described below relate to amplification using polymerase chain reaction (PCR), many other methods are known in the art for amplifying nucleic acids (e.g., isothermal, rolling circle, etc.). It is understood by those skilled in the art that these other methods may be used in place of or in conjunction with PCR methods. See, e.g., Saiki, “Amplification of Genomic DNA” in PCRProtocols, Innis et al., Eds., Academic Press, San Diego, CA 1990, pp. 13-20; Wharam et al., Nucleic Acids Res., 29(11):E54-E54, 2001; Hafner et al., Biotechniques, 30(4):852-56, 858, 860, 2001; Zhong et al., Biotechniques, 30(4):852-6, 858, 860, 2001.

[0039] As used in this paper, the term "detection" refers to observing a signal from a detectable marker to indicate the presence of a target. More specifically, detection is used to detect the context of a specific sequence.

[0040] The terms “complementary,” “complementary,” or “complementarity” as used herein with respect to polynucleotides (i.e., sequences of nucleotides, such as oligonucleotides or genomic nucleic acids) are related to the base pairing rules. As used herein, complementarity of a nucleic acid sequence means that the oligonucleotides (where, when aligned with a nucleic acid sequence, the 5′ end of one sequence pairs with the 3′ end of another) are in “antiparallel binding.” For example, the sequence 5′-AGT-3′ is complementary to the sequence 3′-TCA-5′. Certain bases not typically found in native nucleic acids may be included in the nucleic acids disclosed herein, including, for example, hypoxanthine and 7-guanine. Complementarity does not need to be perfect; a stable double helix may contain mismatched base pairs or unmatched bases. Considering several variable factors, including, for example, the length of the oligonucleotide, the base composition and sequence of the oligonucleotide, the ionic strength and occurrence of mismatched base pairs, those skilled in the art of nucleic acid technology can empirically determine the stability of the double helix. Complementarity can be “partial,” where only some nucleic acid bases match according to the base pairing rules. Alternatively, it could be "complete," "full," or "complete" complementarity between nucleic acids.

[0041] As used herein, the term "detectable marker" refers to a molecule or compound or group of molecules or compounds that is associated with a probe and is used to identify a probe that hybridizes with a genomic nucleic acid or a reference nucleic acid.

[0042] In the context of polynucleotides, a “fragment” refers to a sequence of nucleotide residues, double-stranded or single-stranded, consisting of at least about 2 nucleotides, at least about 5 nucleotides, at least about 10 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, or at least about 100 nucleotides.

[0043] The term "identity" or "identical" refers to the degree of similarity between sequences. There can be partial or complete identity. A partially identical sequence is one that is less than 100% identical to another sequence. Partially identical sequences can have at least 70% or at least 75%, at least 80% or at least 85%, or at least 90% or at least 95% complete identity.

[0044] As used herein, the terms “isolated,” “purified,” or “basically purified” refer to molecules, such as nucleic acids, that have been removed from their natural environment, are isolated or separated, and are free from at least 60%, 70%, 80%, 90%, or 95% of other components naturally associated with them. Isolated molecules are therefore essentially purified molecules.

[0045] As used herein, the term "library" refers to a collection of nucleic acid sequences, such as those derived from the whole genome, subgenomic fragments, cDNA, cDNA fragments, RNA, RNA fragments, or combinations thereof. In one embodiment, some or all of the library's nucleic acid sequences contain adaptor sequences. Adaptor sequences may be located at one end or both ends. Adaptor sequences can be used, for example, by sequencing methods (e.g., NGS), amplification, reverse transcription, or cloning into a vector.

[0046] The library may contain a collection of nucleic acid sequences, such as target nucleic acid sequences (e.g., tumor nucleic acid sequences), reference nucleic acid sequences, or combinations thereof. In some embodiments, the nucleic acid sequences of the library are derived from a single subject. In other embodiments, the library may contain nucleic acid sequences from more than one subject (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or more subjects). In some embodiments, two or more libraries from different subjects may be combined to form a library having nucleic acid sequences from more than one subject. In one embodiment, the subject is a human with cancer or tumor, or at risk of having cancer or tumor.

[0047] "Library nucleic acid sequence" refers to a nucleic acid molecule, such as DNA, RNA, or a combination thereof, that is a member of a library. Generally, the library nucleic acid sequence is a DNA molecule, such as genomic DNA or cDNA. In some embodiments, the library nucleic acid sequence is fragmented, such as spliced ​​or enzymatically prepared genomic DNA. In some embodiments, the library nucleic acid sequence contains sequences derived from the subject and sequences not derived from the subject, such as adaptor sequences, primer sequences, or other sequences that allow recognition of sequences such as "barcodes."

[0048] As used in this article, the term "multiplex PCR" refers to the amplification of two or more target nucleic acids, each initiated using different primer pairs.

[0049] As used herein, “next-generation sequencing or NGS” refers to any sequencing method that determines the nucleotide sequence of a single nucleic acid molecule (e.g., in single-molecule sequencing) or a clonal amplification representative of a single nucleic acid molecule in a high-throughput parallel manner (e.g., automated sequencing of more than 10³, 10⁴, 10⁵ or more molecules). In one embodiment, the relative abundance of nucleic acid species in a library can be estimated by counting the relative number of homologous sequences occurring in the data generated by the sequencing experiment. Next-generation sequencing methods are known in the art and are described, for example, in Metzker, M. Nature Biotechnology Reviews 11:31-46 (2010).

[0050] As used herein, the terms “oligonucleotide” or “polynucleotide” refer to short polymers composed of deoxyribonucleotides, ribonucleotides, or combinations thereof. Oligonucleotides are typically about 10, 11, 12, 13, 14, 15, 20, 25, or 30 to about 150 nucleotides (nt) in length, more preferably about 10, 11, 12, 13, 14, 15, 20, 25, or 30 to about 70 nt.

[0051] As used herein, a "primer" is an oligonucleotide that is complementary to the target nucleotide sequence and, in the presence of DNA or RNA polymerase, causes the addition of nucleotides to the 3' end of the primer. The 3' end of the primer should generally be identical to the target sequence at the corresponding nucleotide position for optimal elongation and / or amplification. The term "primer" encompasses all forms of primers, which can be synthetic, including peptide nucleic acid primers, locked nucleic acid primers, phosphate-thioester modified primers, labeled primers, etc. As used herein, a "forward primer" is a primer complementary to the antisense strand of DNA. A "reverse primer" is complementary to the sense strand of DNA.

[0052] Hybridization refers to the process by which a nucleic acid-specific oligonucleotide (e.g., a probe or primer) "hybridizes" with a target nucleic acid under appropriate conditions. As used herein, "hybridization" refers to the process by which a single strand of an oligonucleotide anneals to its complementary strand through base pairing under defined hybridization conditions. It is a specific (i.e., non-random) interaction between two complementary polynucleotides. The strength of hybridization and the intensity of the hybridization (i.e., the strength of the connection between nucleic acids) are influenced by factors such as the degree of complementarity between the nucleic acids, the strictness of the conditions involved, and the Tm of the resulting hybrid.

[0053] "Specific hybridization" is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissible annealing conditions and remain hybridized after any subsequent washing step. Permissible annealing conditions for nucleic acid sequences can generally be determined by those skilled in the art and can occur, for example, at 65ºC in the presence of approximately 6×SSC. Partially, the stringency of hybridization can be expressed according to the temperature at which the washing step is performed. Such temperatures are typically chosen to be approximately 5ºC to 20ºC lower than the thermal melting point (Tm) of the specific sequence at a given ionic strength and pH. Tm is the temperature at which 50% of the target sequence hybridizes with a perfectly matched probe (at a given ionic strength and Tm). Equations for calculating Tm and the conditions for nucleic acid hybridization are known in the art.

[0054] As used herein, an oligonucleotide is "specific" to nucleic acids if it hybridizes to the target of interest and substantially does not hybridize to nucleic acids of no interest. A high level of sequence identity is preferred and includes at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity. Sequence identity can be determined using a commercially available computer program (e.g., BLAST) with default settings employing algorithms well-known in the art.

[0055] The term "region of interest" refers to the region of nucleic acid that is being sequenced.

[0056] As used herein, the term "subject" refers to a mammal, such as a human, but can also refer to other animals, such as domesticated animals (e.g., dogs, cats, etc.), livestock (e.g., cattle, sheep, pigs, horses, etc.), or laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.). The term "subject" may be used interchangeably with "individual" or "patient" in this document.

[0057] Overview

[0058] The detection and quantification of fetal nucleic acids in mammalian plasma is a non-invasive alternative for prenatal genetic diagnosis. Effective diagnosis depends on the quantity and quality of isolated DNA, which affects the sensitivity and reproducibility of the assay. This paper describes methods for improving the quantity and quality of cell-free nucleic acids (e.g., cell-free, cfDNA) isolated from liquid biological samples. The methods presented herein also reduce inter-sample variability in isolated cfDNA (i.e., improve the consistency of the cfDNA extraction process). The methods presented herein improve the accuracy of fetal fractionation analysis and the detection of fetal aneuploidy. The methods presented herein can also be applied to the isolation and analysis of other forms of cell-free DNA, including circulating tumor DNA.

[0059] Cell-free nucleic acids can be isolated from any type of suitable liquid biological sample or specimen (e.g., a test sample). A sample or test specimen can be any sample isolated from or obtained from a subject or a part thereof (e.g., a human subject, a pregnant female, a fetus). Non-limiting examples of samples include fluids from the subject, including but not limited to, blood or blood products (e.g., serum, plasma, etc.), umbilical cord blood, amniotic fluid, cerebrospinal fluid, cerebrospinal fluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, duct, ear, arthroscopy), lavage fluid from the female reproductive tract, urine, feces, sputum, saliva, nasal mucus, prostatic fluid, lavage fluid, semen, lymph, bile, tears, sweat, breast milk, milk, etc., or combinations thereof. As used herein, the term "blood" refers to a blood sample or preparation from a pregnant woman or a woman undergoing a test for possible pregnancy. The term covers whole blood, blood products, or any fraction of blood, such as serum, plasma, erythrocyte sedimentation rate (ESR), etc., as conventionally defined. Blood or fractions thereof often contain nucleosomes (e.g., maternal and / or fetal nucleosomes). Nucleosomes contain nucleic acids and are sometimes cell-free or intracellular. Blood also contains an erythrocyte sedimentation rate (ESR) layer. Sometimes the ESR layer is separated using a sucrose gradient. The ESR layer may contain white blood cells (e.g., leukocytes, T cells, B cells, platelets, etc.). In some embodiments, the ESR layer contains maternal and / or fetal nucleic acids. Blood plasma refers to the fraction of whole blood obtained by centrifuging blood treated with an anticoagulant. Blood serum refers to the aqueous portion of the liquid remaining after a blood sample has clotted. Liquid samples are often collected according to standard protocols typically followed in hospitals or outpatient clinics. For blood, a suitable amount of peripheral blood (e.g., 3-40 ml) is often collected and stored according to standard procedures before or after preparation. Liquid samples from which nucleic acids have been extracted may be cell-free (e.g., cell-free). In some embodiments, liquid or tissue samples may contain cellular elements or cellular remnants. In some implementations, cellular elements or cellular residues are removed from the liquid sample prior to nucleic acid extraction.

[0060] Samples are often heterogeneous, meaning that more than one nucleic acid species are present in the sample. For example, heterogeneous nucleic acids may include, but are not limited to, (i) fetal and maternal nucleic acids, (ii) cancer or non-cancer nucleic acids, (iii) pathogen or host nucleic acids, or (iv) mutant and wild-type nucleic acids.

[0061] For the prenatal application of the techniques described herein, liquid samples may be collected from females of appropriate gestational age for testing, or from females undergoing testing for possible pregnancy. Appropriate gestational age may vary depending on the prenatal test being performed. In some embodiments, the pregnant female subject may be in the first trimester, the second trimester, or sometimes the last trimester. In some embodiments, the fluid sample is collected from pregnant females with fetal pregnancies of approximately 1 to approximately 45 weeks (e.g., fetal pregnancies of 1-4, 4-8, 8-12, 12-16, 16-20, 20-24, 24-28, 28-32, 32-36, 36-40, or 40-44 weeks), or with fetal pregnancies of approximately 5 to approximately 28 weeks (e.g., fetal pregnancies of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 weeks). In some embodiments, the fluid sample is collected from pregnant females during or immediately after delivery (e.g., vaginal or non-vaginal delivery, such as cesarean section) or immediately after delivery (e.g., 0-72 hours later).

[0062] Methods for preparing serum or plasma from maternal blood are known. For example, blood from a pregnant woman can be placed in a tube containing EDTA or a specialized commercial product such as a Streck BCT collection tube (Streck), which stabilizes leukocytes, prevents the release of genomic DNA, and allows for the separation of high-quality cell-free DNA. Plasma can then be obtained from whole blood by centrifugation. Serum can be obtained with or without blood clotting after centrifugation. If centrifugation is used, it is usually, but not exclusively, carried out at an appropriate speed, such as 1,500–3,000 g. Plasma or serum may undergo an additional centrifugation step before being transferred to a new test tube for cfDNA extraction.

[0063] In some embodiments, the methods described herein can be applied to the isolation of cell-free DNA for the detection of circulating tumor DNA. Therefore, in some embodiments, the subject is a subject diagnosed with or suspected of having cancer.

[0064] Methods for extracting cell-free DNA

[0065] The method described in this paper produces high-quality and high-yield cfDNA samples and reduces inter-sample variability in extracted cfDNA.

[0066] In the first step of this method, a liquid biological sample containing cfDNA is contacted with multiple magnetic solid particles, such as magnetic beads having a surface containing one or more functional groups that reversibly bind DNA. In some embodiments, the magnetic beads are coated with a silicon-based coating. In some embodiments, the magnetic beads are coated with a compound having functional groups that reversibly bind DNA.

[0067] Any solid surface that can bind DNA and has sufficient surface area to allow for effective binding can be used in the methods of the present invention. Microparticles, fibers, beads, and supports contain suitable surfaces. Generally, magnetic beads are used in the methods of the present invention. In some embodiments, the magnetic microparticles used in the methods of the present invention comprise a magnetic metal oxide core surrounded by an adsorbently or covalently bonded silane coating, wherein a variety of biocompatible adsorbents can be selectively coupled chemically covalently bonded, thereby coating the surface of the microparticles with functional groups. In some embodiments, the magnetic beads comprise a polymer. In some embodiments, the polymer is cross-linked polystyrene. In some embodiments, the surface of the magnetic beads is coated with a silicon-containing coating. In some embodiments, the magnetic beads comprise a polymer core, a magnetic layer comprising a magnetic metal oxide covering the polymer, and a silicon-containing outer layer covering the magnetic layer. In some embodiments, the magnetic beads are of uniform size. In some embodiments, the diameter of the magnetic beads is about 1 µm.

[0068] In some embodiments, the magnetic metal oxide is iron oxide. In some embodiments, the iron oxide is Fe. 2+ and Fe 3+ A mixture. Fe 2+ / Fe 3+ The ratio can be changed from about 0.5:1 to about 4:1, such as, for example, 2:1. Suitable aminosilanes for coating the surface of microparticles include, but are not limited to, p-propyltrimethoxysilane, N-2-aminoethyl-3-aminopropyltrimethoxysilane, triaminofunctional silanes (H2NCH2--NH--CH2CH2--NH--CH2--Si--(OCH3)3), n-dodecyltriethoxysilane, and n-hexyltrimethoxysilane. Methods for preparing these microparticles are described in U.S. Patent Nos. 4,628,037, 4,554,088, 4,672,040, 4,695,393, and 4,698,302, the teachings of which are hereby incorporated herein by reference in their entirety. These patents disclose other aminosilanes suitable for coating iron oxide cores or layers, and which can be used in the methods of the present invention.

[0069] In some embodiments, the volume of the liquid biological sample is approximately 0.5-2 mL. In some embodiments, a suitable binding buffer is added to the liquid biological sample before, after, or simultaneously with contact with the magnetic solid particles. In some embodiments, the binding buffer contains a combination of a dissociating salt and a detergent that effectively releases cell-free DNA from any associated histones. Examples include, but are not limited to, guanidine (iso)thiocyanate, guanidine hydrochloride, sodium iodide, potassium iodide, sodium (iso)thiocyanate, urea, or combinations thereof. In a particular embodiment, the dissociating salt is guanidine (iso)thiocyanate.

[0070] Liquid biological samples and magnetic solid-phase particles are incubated for a sufficient time to bind cfDNA in the liquid biological sample to generate cfDNA-bound magnetic solid-phase particles. The cfDNA-bound magnetic solid-phase particles are then separated from the unbound components of the liquid sample by transferring the magnetic particles to a container containing a suitable washing solution. The washing buffer solution must have a sufficiently high salt concentration (i.e., a sufficiently high ionic strength) so that the DNA bound to the magnetic particles does not elute the particles but remains bound. A suitable salt concentration is above about 1.0 M, and preferably about 5.0 M. A buffer solution is selected to dissolve the impurities that bind the DNA or the particles. The pH, solute composition, and concentration of the buffer solution can be varied depending on the type of impurities expected to be present. Suitable washing solutions include: a washing buffer containing guanidine salt, 0.5 × 5 SSC; 100 mM ammonium sulfate, 400 mM Tris pH 9, 25 mM MgCl2, and 1% fetal bovine serum albumin (BSA); and 5 M NaCl. Magnetic particles and bound DNA can also be washed with more than one washing buffer solution. Magnetic microparticles can be washed frequently as needed to remove desired impurities. However, the number of washes is preferably limited to two or three times to minimize the loss of bound DNA.

[0071] In some embodiments, two or more washing steps are performed by transferring the magnetic particles to an additional container containing a washing solution for each additional wash. The washed cfDNA-bound magnetic solid particles are then transferred from the washing solution to a container containing an ethanol washing solution. In some embodiments, the ethanol washing solution is a 70%, 75%, 80%, 85%, or 90% ethanol solution. In some embodiments, two or more ethanol washing steps are performed by transferring the magnetic particles to an additional container containing an ethanol washing solution for each additional ethanol wash. The ethanol-washed cfDNA-bound magnetic solid particles are then transferred to a container containing an elution solution. A suitable elution solution releases the bound cfDNA from the magnetic beads. Exemplary elution solutions include water or buffer solutions such as Tris buffer (e.g., 10 mM Tris, pH 7.0–8.0) or TRIS-EDTA buffer.

[0072] In some embodiments, cfDNA-bound magnetic solid particles are magnetically bound to a liquid sample containing such particles by sequentially inserting vertical rod-shaped magnets into the sample. The magnetically bound particles are then transferred to a container containing multiple wash and elution solutions, and deposited in a subsequent container containing a wash or elution solution. An exemplary vertical rod-shaped magnet used in this invention is described in U.S. Patent 6,447,729. Generally, the rod-shaped magnet is protected with a plastic tip (e.g., polypropylene) having low / no binding affinity for biomolecules. In some embodiments, the tip is shaped to match a porous plate (e.g., a 24-well magnetic tip comb used with a 24-well plate, Kingfisher). This allows the magnet to retract from the tip, leaving the tip in the pore, which allows the magnetic particles to be released into the receiving solution with low agitation. Once the magnetic particles have been released, the plastic tip can be removed from the pore. Alternatively, the plastic tip can remain in the pore during mixing. The magnetism of the rod is reused to transfer the magnetic particles to a subsequent container.

[0073] The separation of magnetic solid particles from the elution solution containing eluted cfDNA is accomplished by transferring the container containing magnetic beads in the elution solution to a magnetic plate, such as a magnetic plate containing ring magnets, and the removal of the cfDNA-containing elution solution. The cfDNA-containing elution solution is then removed from the container, leaving the magnetic solid particles. In some embodiments, heat is applied to the magnetic solid particles to elute the cfDNA.

[0074] In some embodiments, the volume of the elution solution is less than 200 µl. In some embodiments, the volume of the elution solution is less than or about 150 µl, less than or about 100 µl, less than or about 90 µl, less than or about 80 µl, less than or about 70 µl, less than or about 60 µl, less than or about 50 µl, or less than or about 40 µl. In some embodiments, the volume of the elution solution is about 70 µl.

[0075] In some implementations, the cfDNA extraction method uses a multi-well plate. In some implementations, the multi-well plate is a deep-well plate. In some implementations, the multi-well plate is a 24-well deep-well plate.

[0076] One advantage of the method of the present invention is that multiple aliquots of liquid biological samples can be contacted with magnetic solid-phase particles, and the magnetic solid-phase particles bound to cfDNA from each binding reaction can be assembled prior to washing. For example, a vertical rod magnet can be used to collect the magnetic solid-phase particles bound to cfDNA from each binding reaction before all the cfDNA-bound particles are deposited into a first washing solution. Thus, in some embodiments, at least two replicate liquid biological samples containing cfDNA are contacted with multiple magnetic particles. In some embodiments, at least two or more replicate liquid biological samples containing cfDNA are contacted with multiple magnetic particles. Such a method improves the efficiency of the separation step and the yield of extracted cfDNA. This method differs from previous methods in which aliquots of liquid biological samples are treated separately or sequentially applied to magnetic solid-phase particles for binding. The method of the present invention allows for simultaneous binding and aggregation of samples.

[0077] Another advantage of the method of the present invention is that the application of a magnetic plate to the elution solution containing magnetic solid particles improves the final yield and allows for the use of lower elution volumes to separate the magnetic particles from the eluted cfDNA. In previous methods using vertical rod magnets for DNA extraction, the vertical rod magnets were used to transfer the magnetic solid particles to the wash solution and to remove the magnetic solid particles from the elution solution in the final elution step (see, for example, KingFisher® Flex System (Thermo Scientific)). To achieve acceptable and consistent yields from such systems, higher elution volumes (500 µl) are required, especially when using deep-well multi-well plates (e.g., 24-well deep-well plates) (see www.thermo.com / kingfisher). Lower elution volumes result in irreversible or insufficiently eluted DNA. Lower elution volumes require a reduced tip height to contact the elution solution, which results in random beads breaking through the plate during mixing. It was found here that, after removing the beads, the method results in at least 5-10% of wells containing irreversible or insufficiently eluted DNA. Conversely, when using low elution volumes (e.g., less than 200 µl), the method of the present invention, using a plate magnet for the elution step, produces high yields and high-quality cfDNA with low inter-sample variability. Lower elution volumes reduce the need to concentrate samples for downstream library preparation and / or direct sequencing steps.

[0078] In some implementations, the DNA extraction step is automated. For example, a robotic arm is used to transfer magnetic beads from one container to another. In some instances, instruments equipped with vertical rod-shaped magnets, such as the KingFisher™ Flex Magnetic Particle Processor (Thermo Scientific, Waltham, MA), are used to collect cfDNA-bound magnetic particles from liquid biological samples and to deposit the beads in a washing or elution solution.

[0079] Library generation and sequencing

[0080] In some implementations, the extracted cfDNA is used to prepare an amplified library for high-throughput sequencing. Generally, cfDNA does not need to be fragmented. In some implementations, the cfDNA size ranges from approximately 150 bp to 200 bp. In some implementations, the average cfDNA size is from approximately 170 bp to approximately 175 bp.

[0081] In an exemplary method, cell-free DNA is first blunted and 5'-phosphorylated, allowing the cfDNA fragment to be ligated to an adaptor. This process can be performed using commercially available kits such as the NEB Quick Blunting Kit® or NEBNextEnd Prep.

[0082] The prepared cfDNA can then be ligated to a nucleic acid adaptor, which allows for nucleic acid amplification, target labeling, and / or sequencing. In some embodiments, the first adaptor ligates the 5' end and / or 3' end of the prepared cfDNA fragment. In a particular embodiment, the first adaptor is a Y-shaped adaptor containing two partially complementary oligonucleotides. Such an adaptor allows for differential labeling.

[0083] In some implementations, the "positive" Y-shaped connector sequence consists of or contains the following sequences: The sequence is 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:1, and the “reverse” Y-shaped connecting subsequence consists of or contains the following sequences: Or a sequence that is 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 2. The "forward" sequence... and in the "reverse" sequence It is a phosphate thioester bond. The underlined sequence represents the complementary nucleotide sequence in each oligonucleotide. The neck (i.e., the double-stranded portion) of the Y-shaped adaptor end has a T-hang, which allows the cfDNA fragment to be ligated with blunt-end / TA ligase (NEB). Depending on the manufacturer's protocol, the adaptor ligation reaction can be purified using commercially available kits such as Ampure® XP PCR purification beads (Beckman Coulter, #A63881) to remove unligated adaptors and other impurities.

[0084] In some implementations, the product ligated to the adaptor is then amplified using an amplification primer pair, wherein one amplification primer in the primer pair is a universal primer, and the other primer in the primer pair is a barcoded reverse primer. The universal primer and / or the barcoded primer provide a start site for sequencing. This sequence allows the fragment to be combined with a flow cell for high-throughput, massively parallel sequencing, as described herein.

[0085] In some cases, amplicons from a single sample source contain the same index sequence (also known as an index tag, "barcode," or multiple identifier (MID)). In other cases, indexed amplicons are generated using primers containing the index sequence (e.g., forward and / or reverse primers). Such indexed primers can be included during library preparation as a "barcode-adding" tool to identify specific amplicons originating from a particular sample source. Amplicons from indexed sources from more than one sample source are quantified individually and then pooled before sequencing. Therefore, the use of index sequences allows for the pooling of multiple samples (i.e., samples from more than one sample source) per sequencing run, and the sample source is subsequently determined based on the index sequence.

[0086] Exemplary general-purpose forward primers and barcode-encoded reverse primers allow the following primers:

[0087] P5 General:

[0088] MID primers: , where “NNNNNNNNNN” represents the molecular barcode sequence.

[0089] Any linker sequence may be included in the primers used in this invention.

[0090] In some implementations, all forward amplicones (i.e., amplicones extending from a forward primer that hybridizes to the antisense strand of the target region) contain the same adaptor sequence. In some implementations, when double-stranded sequencing is performed, all forward amplicones contain the same adaptor sequence, and all reverse amplicones (i.e., amplicones extending from a reverse primer that hybridizes to the sense strand of the target region) contain an adaptor sequence different from that of the forward amplicones.

[0091] Other adaptor sequences are known in the art. Some manufacturers recommend specific adaptor sequences for use with specific sequencing technologies and the machines they provide.

[0092] In some implementations, when adaptor-linked and / or indexed primers are used to amplify cfDNA fragments, the adaptor sequence and / or index sequence are incorporated into the amplicon during amplification (along the target-specific primer sequence). The presence of the index tag allows for sequence variability from multiple sample sources.

[0093] In some implementations, amplicon libraries from more than one sample source are pooled prior to high-throughput sequencing. "Compounding" involves pooling libraries with multiple adaptor tags and indexes into a single sequencing run. This capability can be used for comparative studies when using indexed primer sets. In some implementations, amplicon libraries from up to 48 independent sources are pooled prior to sequencing.

[0094] High-throughput, massively parallel sequencing refers to sequencing methods that can generate multiple sequencing reactions in parallel to produce cloned amplified molecules and single nucleic acid molecules. This allows for increased throughput and data generation. These methods are also known in the art as next-generation sequencing (NGS) methods. NGS methods include, for example, sequencing-by-synthesis using reversible dye terminators and sequencing-by-ligation.

[0095] In some implementations, high-throughput, massively parallel sequencing employs sequencing by synthesis using reversible dye terminators. In other implementations, sequencing is performed via ligation sequencing. In yet another implementation, sequencing is single-molecule sequencing.

[0096] Non-limiting examples of commonly used NGS platforms include the Apollo 324™ NGS Library PrepSystem (IntengenX, Pleasanton, United States), Ion Torrent™ (LifeTechnologies, Carlsbad, CA), miRNA BeadArray (Illumina, Inc.), Roche 454™ GSFLX™-Titanium (Roche Molecular Diagnostics, Germany), and ABI SOLiD™ System (Applied Biosystems, Foster City, CA). After generating adaptor tags and optionally indexing amplicon libraries, the amplicon sequences are sequenced using high-throughput, massively parallel sequencing.

[0097] Determination of fraction of acellular fetus

[0098] Determining the fetal fraction in male fetuses is relatively straightforward because the amount of Y chromosome present, and the observed normalized X chromosome read count defects relative to female fetal samples, can be used to directly calculate the fetal fraction. Determining the presence of fetal DNA from female fetuses is more difficult. This requires analysis of autosomes to confirm its presence. Specific loci on autosomes show an increase in cfDNA of fetal origin. The degree of enrichment at these loci can be used to determine the presence of female fetal DNA.

[0099] Sequencing data generated by NGS methods on cfDNA fragment libraries can be analyzed to determine the cell-free fetal fraction, which distinguishes the portions of cfDNA attributed to fetal DNA from those attributable to maternal DNA. Exemplary methods for determining the fetal fraction include measuring the relative levels of fetal-specific genes, maternal-specific genes, and / or male-specific genes. Once the cell-free fetal fraction is determined, the presence of a specific aneuploidy can be estimated. Exemplary methods for determining aneuploidy from NGS data are described, for example, in US2015 / 0005176. Sequencing reads are aligned to a reference human genome using a short-read aligner. Aneuploidy is defined as the number of reads mapped to each chromosome differing from its expected number, based on comparison with a set of baseline samples. A common approach is often referred to as a read depth strategy. In cfDNA testing, the counts of specific cfDNA fragments from a specific chromosome are compared in euploid and trisomic pregnancies, and the results are expressed as Z-scores. The intervals between the distributions of Z-scores from euploid and trisomic pregnancies increase with deeper sequencing levels and increasing fetal fractions. As illustrated in the embodiments provided herein, cfDNA extraction according to the provided method results in higher fetal scores, and therefore, higher Z scores for accurate and sensitive prediction of fetal aneuploidy.

[0100] Example

[0101] Example 1: Cell-free DNA extraction via NIPT cycles

[0102] Whole blood from the intended female contains both maternal cell-free DNA (cfDNA) and fetal cfDNA. Whole blood is collected from the intended mother in specific blood collection tubes to prevent maternal cell lysis and the release of their DNA into the plasma. Preserving maternal cells and preventing their DNA from entering the plasma prevents the ratio of fetal cfDNA to maternal DNA from dropping to a point where fetal cfDNA might be undetectable. When the blood is obtained in the laboratory, it is centrifuged to separate the plasma, erythrocyte sedimentation rate (ESR) layer, and red blood cell fraction. cfDNA (maternal and fetal) is then extracted from the plasma fraction of the whole blood.

[0103] The methods described herein are used to isolate circulating cfDNA from maternal plasma samples using magnetic beads (e.g., DynabeadsMyOne Silane) that bind DNA to the sample. The extracted DNA is used for downstream sequencing applications, such as tests for the detection of specific chromosomal abnormalities.

[0104] Dynabeads® MyOne™ SILANE are uniform, single-sized ferromagnetic beads with a diameter of 1 μm. The beads consist of highly cross-linked polystyrene with a uniformly distributed magnetic material. The beads are further coated, encapsulating iron oxide within the beads and exhibiting an optimized silica-like chemistry on the surface. The increased magnetic strength of these beads ensures rapid magnetic mobility and efficient separation of nucleic acids (DNA / RNA). The beads also feature low sedimentation rates and favorable reaction kinetics, making them particularly suitable for automated assays (see Product Description for Dynabeads® MyOne™ SILANE, Life Technologies).

[0105] Each maternal blood sample was collected in two Streck BCT collection tubes and stored at ambient temperature for up to 4 days. Plasma was then separated twice by spin sampling. Reagent and plasma sample plates were first prepared on a Hamilton Microlab STAR liquid processor (Hamilton Company, Reno, NV). The prepared plates, along with a comb plate, were then loaded onto a KingFisher™ Flex magnetic particle processor (with 24 deep-well heads) (Thermo Scientific, Waltham, MA) for DNA extraction. Circulating DNA was efficiently released at room temperature from histones coated with a unique combination of dissociative salts and detergent.

[0106] Free dsDNA binds to Silane magnetic beads, which are then collected on a comb with a magnetic rod inserted into the center. After several washes, non-specific binding proteins are removed. The Silane beads are released from the comb, and the dsDNA is eluted in DNA elution buffer. Finally, the KingFisher™ DNA elution plate, still containing the magnetic beads in the elution buffer, is transferred to a magnetic plate containing a ring magnet on a Hamilton Microlab Starlet. This transfers the eluted DNA solution to a DNA collection plate, which is then stored at -20°C until use. Detailed instructions for the DNA extraction protocol are provided below.

[0107] plan

[0108] Whole blood samples (approximately 20 mL) from patients are collected in Streck BCT collection tubes (Streck, Ct#218962, approximately 10 mL per tube, two tubes per patient). Samples are transported to the processing laboratory at ambient temperature (18–26°C). For best results, samples should be received at the laboratory within 4 days of collection from the patient.

[0109] The Streck tubes were centrifuged at 2,500 × g (RCF) for 10 minutes at 22°C in a round-bottom barrel adapter. After centrifugation, the Streck tubes were transferred to a safe fume hood without disturbing the erythrocyte sedimentation rate (ESR) brown layer. Plasma was collected from each tube without contact with the ESR brown layer. The plasma from each patient's two Streck tubes was pooled into the first conical tube (tube #1). After removing the plasma, the Streck tubes were recapped with their original stoppers, and the remaining material was stored at 4°C.

[0110] Centrifuge the conical tubes at 3,200 × g (RC) for 20 minutes at 22°C in a conical-bottom barrel adapter. After centrifugation, collect the plasma. Transfer 4.5 mL of the collected plasma to a second conical tube (tube #2), and transfer the remaining collected plasma to a third conical tube (tube #3) for later use. Discard the remaining cell pellet in conical tube #1. For same-day DNA extraction, store plasma tube #2 at 4°C. For long-term storage, store this tube at -20°C or lower until use.

[0111] If the plasma is still frozen before DNA extraction, thaw it at room temperature. Once thawed, centrifuge the plasma tubes at 3,000 × g (RCF) for 10 minutes. Record any tubes containing less than 3 mL of plasma.

[0112] Then prepare the Dynabeads binding solution stock. Remove the container of Dynabeads® MyOne™ SILANE (LifeTechnologies – Catalog No. 37002D) from the refrigerator and vortex it vigorously for 1 minute just before adding the DynaMax binding solution (DynaMax Cellless DNA Extraction Kit, Catalog No. 4479983). Add 1500 µL of Dynabeads® MyOne™ SILANE magnetic beads to 125 mL of DynaMax binding solution (DynaMax Cellless DNA Fun Kit, Catalog No. 4479983) to prepare the Dynabeads binding solution stock.

[0113] The 24-well plates (KingFisher™ Flex 24-well plates, Molecular BioProducts Inc., catalog number 95040480) of the lower group were prepared using a Hamilton Microlab STAR liquid processor (Hamilton Company, Reno, NV).

[0114] 1) 2 mL of patient plasma sample plus 2 mL of Dynabeads binding solution stock (prepared above) (total volume 4 mL).

[0115] 2) Two copies of plate #1.

[0116] 3) 1 mL DynaMax washing solution (DynaMax Cell-Free DNA Extraction Kit, Catalog No. 4479983)

[0117] 4) 1 mL DynaMax washing solution (DynaMax Cell-Free DNA Extraction Kit, Catalog No. 4479983)

[0118] 5) 2 mL 80% ethanol

[0119] 6) 600 µl 80% ethanol

[0120] 7) DynaMax elution solution (DynaMax cell-free DNA extraction kit, catalog number 4479983)

[0121] The prepared plate was then unloaded from the Hamilton Microlab STAR, and reagent volume consistency was checked. The prepared plate was then loaded onto a KingFisher™ Flex magnetic particle processor (with 24 deep-well heads) (Thermo Scientific, Waltham, MA). A plate containing a KingFisher™ Flex 24-well comb (Molecular BioProducts Inc. – Catalog No. 97002610) was loaded onto the KingFisher™ processor. In this individual step, the plate was held stationary, and the only moving component was the processing head with the comb and magnetic rod. This head consisted of two vertically moving platforms. One platform moved the plastic comb in and out of the deep-well sample plate, and the other platform moved the magnetic rod in and out of the plastic comb. The operating principle used for DNA extraction is called inverse magnetic particle processing (MPP) technology. Instead of moving liquid in and out of the plate, as is done with external magnets, the magnetic particles themselves are moved from the plate to the plate containing the specific reagent. Magnetic particles are transferred to each consecutive plate (#1-#7) with the aid of magnetic rods covered with disposable, specially designed plastic tips.

[0122] The procedure for isolating cfDNA from plasma samples is as follows: 1) Collect cfDNA bound to magnetic particles on plate #1 (i.e., DNA-bound magnetic particles are bound to the comb by inserting the comb and magnetic rod assembly into the wells of plate #1); 2) Collect cfDNA bound to magnetic particles on plate #2 (i.e., DNA bound to magnetic particles on plate #2 also becomes bound to the comb); 3) Wash first with DynaMax washing solution; 4) Wash second with DynaMax washing solution; 5) Wash first with 80% ethanol; 6) Wash second with 80% ethanol; and 7) Elute with 70 µl of DynaMax elution solution. Between each step, beads are released from the comb by removing the magnetic rod from the comb assembly and re-bound by inserting the magnetic rod back into the comb assembly.

[0123] Under standard operating conditions, as beads are deposited in the elution buffer, the KingFisher™ processor removes the beads from the elution buffer during step 7, leaving the purified liquid sample containing cfDNA behind. It was found that when using elution buffer volumes smaller than 100 µl, 5–10% of the wells contained unrecoverable or insufficiently eluted DNA. For these wells, a reminder typically yields less than 50 µl of DNA recovered from a 70 µl elution buffer. These poor results may be due to bead collisions occurring during bead deposition and mixing at low elution volumes.

[0124] The current method modifies the standard KingFisher™ DNA purification protocol, where the beads are not removed from the elution solution in step 7, thus allowing for smaller elution volumes. Beads are released into an elution plate (#7) for the elution step, and then the elution plate, still containing beads and elution solution, is transferred to a 24-well magnetic plate with ring magnets. The ring magnets bind the beads to the sides of the wells, thus allowing for more complete removal of the eluted sample. This modified step, using ring magnets to collect the eluted sample, increases the yield of cfDNA in the final sample and allows for much smaller elution volumes. Additionally, the current method results in 100% recovery of at least 65 µl of the 70 µl elution buffer.

[0125] Magnetic plates containing ring magnets are located in the Hamilton Microlab STARlet liquid processor. Once binding of the magnetic beads occurs, the eluted sample containing cfDNA is transferred to a 96-well plate using the liquid processor.

[0126] Example 2: Sequencing of cfDNA samples

[0127] In this embodiment, cfDNA extracted from Example 1 was prepared for sequencing. The extracted DNA was amplified and sample-specific barcodes were attached to the cfDNA fragments. The amplified products were then analyzed using massively parallel sequencing technology (also known as next-generation sequencing (NGS)). The data from the sequencer was then analyzed, and a report on chromosomes 13, 18, 21, X, and Y was generated, indicating the copy number of which chromosomes were present in the fetal sample. This assay can detect Patau syndrome (trisomy 13), Edward syndrome (trisomy 18), Down syndrome (trisomy 21), and sex chromosome abnormalities such as Turner syndrome (X) and Kleinfelter syndrome (XXY).

[0128] plan

[0129] Cell-free DNA was extracted according to the method described in the examples. A library was then prepared from the extracted cfDNA using the Illumina® (New England BioLabs, #E7370B) NEBNext® Ultra™ DNA Library Prep Kit according to the following protocol. For automated reagent aliquoting, a Hamilton Starlet liquid processor was used.

[0130] End repair

[0131] For end repair of cfDNA fragments, prepare the NEBNext End Prep reaction. Thaw the NEBNext End Prep reagent on a frozen block and aliquot it as follows.

[0132] Table 1

[0133]

[0134] Manually aliquot 127 µL of the NEBNext End Prep master mixture into each well of an 8-strip PCR tube on the frozen block. Then, transfer 9.5 µL of the NEBNext End Prep master mixture from the 8-strip tubes to each well of a 96-well plate using a Hamilton Starlet liquid processor. Transfer 55.5 µL of extracted cfDNA to a 96-well PCR plate (total reaction volume 65 µL). Seal the PCR plate with aluminum foil, vortex briefly (approximately 5 seconds), and centrifuge rapidly at 2,000–6,000 rcf (1,600 rpm in a Sorvall T6000D centrifuge or equivalent) for approximately 5–15 seconds.

[0135] The PCR plates were then placed in a thermal cycler and incubated under the following conditions.

[0136] Table 2

[0137]

[0138] Then remove the 96-well plate from the thermal cycler and centrifuge rapidly for about 5-15 seconds in a plate centrifuge at 2,000-6,000 rcf (1,600 rpm in a Sorvall T6000D centrifuge or equivalent).

[0139] Connector Sub-connection

[0140] The Y-shaped NIPT adapter is generated from the following oligonucleotides: NIPT adapter 1 and NIPT adapter 2.

[0141] NIPT connector 1:

[0142] NIPT connector 2:

[0143] The C*T in NIPT adaptor 1 and the A*C in NIPT adaptor 2 are phosphate thioester bonds. The underlined sequences indicate the complementary nucleotide sequences in each oligonucleotide. Primary NIPT adaptors containing hybridized NIPT adaptors 1 and NIPT adaptor 2 were prepared at a concentration of 50 µM according to the table below.

[0144] Table 3

[0145]

[0146] Aliquot 50 µL of the original seed and 50 µL of the adaptor mixture into each well of a new 96-well PCR plate. Seal the plate, place it in a thermal cycler, and incubate under the following conditions:

[0147] Table 4

[0148]

[0149] When the procedure is complete, combine all wells back into a single sterile microcentrifuge tube and vortex 5–10 times. For storage, aliquot 30 μL into sterile microcentrifuge tubes and store at -15°C to -25°C.

[0150] Prepare the linker-connected master mixture according to the following amounts:

[0151] Table 5

[0152]

[0153] Manually aliquot 259 µL of the adapter-ligation master mixture into each well of an 8-strip PCR tube on the frozen block. Then, transfer 18.5 µL of the adapter-ligation master mixture from the 8-strip tube to each well of the end-of-line PCR plate using a Hamilton Starlet liquid processor (total volume = 83.5 µL). Seal the PCR plates with aluminum foil strips, vortex briefly (approximately 5 seconds), and centrifuge rapidly in a plate centrifuge at 2,000–6,000 rcf (1,600 rpm in a Sorvall T6000D centrifuge or equivalent) for approximately 5–15 seconds.

[0154] The PRC plate was then placed in a thermal cycler and incubated under the following conditions.

[0155] Table 6

[0156]

[0157] Then remove the 96-well plate from the thermal cycler and centrifuge rapidly in a plate centrifuge at 2,000–6,000 rcf (1,600 rpm in a Sorvall T6000D centrifuge or equivalent) for about 5–15 seconds.

[0158] Amplure purification of the ligation product

[0159] The adaptor ligation reaction was then purified using Ampure® XP PCR purification beads (Beckman Coulter, #A63881) according to the manufacturer's protocol to remove unligated adaptors and other impurities. The aliquot steps of this method were performed using a Hamilton Starlet liquid processor.

[0160] In short, add 83.5 µL of AMPure XP beads to a ligation product plate, mix, and incubate at room temperature for 10 minutes for DNA binding. Then transfer the plate to a magnetic plate and let it stand for 4 minutes to clarify the supernatant. Remove and discard the supernatant. Add 180 µL of freshly prepared 80% ethanol, incubate for 30 seconds, then remove and discard. Repeat the 80% ethanol wash twice (3 washes in total). After removing the final ethanol wash, air dry the beads on a magnetic plate for 2 minutes. Then remove the plate from the magnet. Add 28 µL of water to resuspend the beads and incubate at room temperature for 2–5 minutes to elute the DNA. Then transfer the plate back to the magnet and let it stand for 2 minutes to clarify the elution solution. Then transfer 23 µL of the supernatant (purified DNA) to a new 96-well PCR plate.

[0161] PCR of the library

[0162] Then, universal forward primers and barcoded reverse primers were used to amplify the linker ligation product.

[0163] P5 universal:

[0164] MID primers:

[0165] (The underlined portion of the MID primer represents a unique 10-nucleotide molecule ID sequence). Exemplary barcode sequences can be found, for example, in US 20140141436 and (Attorney Docket No. 034827-0803).

[0166] The PCR master mixture was prepared according to the following quantities. MID-barcoded primers were aliquoted.

[0167] Table 7

[0168]

[0169] Add 26 µL of the master mixture directly to each well of the purified ligation plate (total volume = 50 µL). Aliquot 2 µL of appropriate MID primers into each well of the PCR plate. Aliquot different barcodes into each well of the plate. Seal the PCR plates with aluminum foil strips, vortex briefly (approximately 5 seconds), and centrifuge rapidly in a plate centrifuge at 2,000–6,000 rcf (1,600 rpm in a Sorvall T6000D centrifuge or equivalent) for approximately 5–15 seconds.

[0170] The PCR plate was then placed in a thermal cycler and incubated under the following conditions.

[0171] Table 8

[0172]

[0173] The 96-well plates were then removed from the thermal cycler and rapidly centrifuged in a plate centrifuge at 2,000–6,000 rcf (1,600 rpm in a Sorvall T6000D centrifuge or equivalent) for approximately 5–15 seconds.

[0174] Purification of library PCR products

[0175] The library PCR reaction was then purified using Ampure® XP PCR purification beads (Beckman Coulter, #A63881) according to the manufacturer's protocol to remove primers and other impurities. The aliquot steps of the method were performed using a Hamilton Starlet liquid processor.

[0176] In short, add 50 µL of AMPure XP beads to a ligation product plate, mix, and incubate at room temperature for 10 minutes for DNA binding. Then transfer the plate to a magnetic plate and let it stand for 4 minutes to clarify the supernatant. Remove and discard the supernatant. Add 180 µL of freshly prepared 80% ethanol, incubate for 30 seconds, then remove and discard. Repeat the 80% ethanol wash twice (3 washes in total). After removing the final ethanol wash, air dry the beads on a magnetic plate for 2 minutes. Then remove the plate from the magnet. Add 33 µL of water to resuspend the beads and incubate at room temperature for 2–5 minutes to elute the DNA. Then transfer the plate back to the magnet and let it stand for 2 minutes to clarify the elution solution. Then transfer 28 µL of the supernatant (purified DNA) to a new 96-well PCR plate.

[0177] The amplified library was quantified using the Quant-It PicoGreen® dsDNA assay kit (Invitrogen, #P7589) according to the manufacturer's protocol. The molar concentration of the library sample was calculated based on an average library size of 298 bp. Therefore, the molecular weight of the library was approximately 193,700 g / mol (298 bp × 650 g / mol per bp).

[0178] After quantitative amplification, the library was normalized to 2 nM with water. The library was pooled through rows of 96-well plates (5 µL / well) to produce 8 pools of 60 μL each for each 96-well plate.

[0179] NGS sequencing

[0180] Then, a normalized pooled library was prepared using the HiSeq SR Cluster Kit v4 cBot, which included high-output single-end flow cells, cBot clustering reagents, and indexing reagents. Flow cell clustering on cBot reagent plates was performed according to the manufacturer's instructions. High-output sequencing was then performed using an Illumina HiSeq 2500 and HiSeq SBS Kit v4 according to the manufacturer's instructions.

[0181] The following control samples were used for each sequencing run:

[0182] 1) PhiX sequencing control

[0183] A low-level peak was observed when 5% of a control library generated from the PhiX virus was added to the sequencing ensemble before cluster generation and loaded onto HiSeq for sequencing. This allowed the Illumina cluster detection algorithm to perform better due to a more balanced expression of A, T, G, and C nucleotides. It also helped identify failed HiSeq runs, whether the failure stemmed from sample preparation or cluster generation in the flow cell. Because the PhiX sequence was known, this peak also helped determine the run's error rate and provided an indication of sequencing success. The phase rate and pre-phase rate were also determined using the PhiX sequence.

[0184] 2) Collected negative plasma controls

[0185] Combine the remaining plasma from previous runs and determine the fetal fraction of the combined mixture. Divide the pooled plasma into single-use portions and freeze until use. Run one of these controls on each 96-well plate and use it as a control for the entire assay protocol. The known fetal fraction used for the control should match the calculated fetal fraction used for each run.

[0186] 3) Positive control

[0187] A positive control was added during the recombination process. This control was generated from the products of the combined library amplification, each with a different barcode attached thereto, including normal euploid samples, with a 5% mixture of each of trisomy 21, trisomy 18 and trisomy 13.

[0188] 4) Negative DNA control

[0189] Negative DNA controls include the following: a) NS (No Sample) Control: Reagent blank, which includes the actual DNA used to extract the sample. If samples are prepared in different settings, a reagent blank control must be included for each individual preparation. b) ND Control: The negative DNA control (ND control) must be placed at the end of the run. This control consists of the PCR kit and polymerase mixture used to determine the run.

[0190] Sequence read alignment and analysis were performed using proprietary software. Z-scores were calculated based on each sample-chromosome pair (i.e., for chromosomes 21, 18, and 13). Exemplary Z-score data, constructed samples, and normal samples from pre-validation studies of known trisomy 21 positivity using cfDNA extraction methods are shown in Table 9.

[0191] Table 9

[0192]

[0193] Table 10 shows exemplary validation results for chromosome 13, 18, and 21 aneuploidy using two quality control metrics: a single read count threshold (i.e., 9 million reads) or a combination of read count and the percentage of reads mapped to the genome.

[0194] Table 10

[0195]

[0196]

[0197] For validation studies, perfect intraassay consistency was observed in at least five replicates of the exemplary negative sample and the exemplary trisomy 21 positive sample.

[0198] Table 11 shows exemplary validation data for Z-scores and fetal scores for multiple pregnancy samples.

[0199] Table 11

[0200]

[0201]

[0202] Table 12 shows exemplary test data for Z-scores used to select positive samples, indicating aneuploidy of chromosomes 13, 18, and 21. The mean Z-scores for wt / wt samples were -0.3 for T13, -0.06 for T18, and -0.06 for T21.

[0203] Table 12

[0204]

[0205] It should be understood that although the invention has been specifically disclosed through preferred embodiments and optional features, modifications, improvements, and alterations of the invention as disclosed herein are possible to those skilled in the art, and such modifications, improvements, and alterations are considered to be within the scope of the invention. The materials, methods, and embodiments provided herein are representative of specific embodiments, are exemplary, and are not intended to be limiting of the scope of the invention.

[0206] The invention is described broadly and generally herein. Each narrower group of species and subgenus falling within the general disclosure also constitutes part of the invention. This includes the general description of the invention, any attached conditions or negative limitations removing any subject matter from that genus, regardless of whether the removed material is specifically enumerated herein.

[0207] Furthermore, when the features or aspects of the invention are described in accordance with the Markush Group, those skilled in the art will recognize that the invention is also described in accordance with any single member or subgroup member of the Markush Group.

[0208] All publications, patent applications, patents and other references mentioned herein are specifically incorporated in their entirety as if they were individually included. In case of conflict, this specification (including definitions) shall prevail.

[0209] The invention described herein by example can be suitably practiced without any one or more elements, or one or more limitations, which are not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” “containing,” etc., should be broadly understood and without limitation. Furthermore, the terminology and expressions used herein have been used as descriptive rather than limiting, and the use of such terminology and expressions is not intended to exclude any equivalents of the features shown and described or portions thereof, but rather to recognize that various modifications are possible within the scope of the claimed invention.

[0210] Further embodiments are illustrated in the following implementation scheme.

[0211] 1. A method for isolating cell-free DNA (cfDNA) from a liquid biological sample derived from a mammal, the method comprising:

[0212] a) Contact a liquid biological sample containing cfDNA with multiple reversibly DNA-binding magnetic beads.

[0213] b) Separate the cfDNA-bound magnetic beads from the unbound components of the liquid biological sample;

[0214] c) Transfer the magnetic beads bound to the cfDNA to a container containing an elution solution;

[0215] d) Separate the magnetic beads from the elution solution and collect the elution solution containing the cfDNA by contacting the container containing the magnetic beads in the elution solution with a magnetic plate.

[0216] 2. The method of embodiment 1, wherein the magnetic beads comprise cross-linked polystyrene.

[0217] 3. The method of embodiment 1, wherein the surface of the magnetic beads contains one or more functional groups that reversibly bind DNA.

[0218] 4. The method of implementation scheme 1, wherein the magnetic beads are coated with a silicon-containing coating.

[0219] 5. The method of embodiment 1, wherein the magnetic beads bound to the cfDNA are separated from the unbound components of the liquid biological sample by transferring the magnetic beads bound to the cfDNA from the liquid biological sample to another container.

[0220] 6. The method of implementation scheme 1, wherein the magnetic beads bound to the cfDNA are directly transferred from the liquid biological sample to a container containing the elution solution.

[0221] 7. The method of embodiment 1, wherein magnetic beads bound to the cfDNA transferred from the liquid biological sample are transferred to a container containing a washing solution.

[0222] 8. The method of embodiment 1, wherein the cfDNA-bound magnetic beads are separated from the unbound components of the liquid biological sample by inserting a rod-shaped magnet into the liquid biological sample to magnetically bind the cfDNA-bound magnetic beads.

[0223] 9. The method of embodiment 8, wherein the magnetic beads are deposited in a container containing the elution solution.

[0224] 10. The method of embodiment 8, wherein the magnetic beads are deposited in a container containing the washing solution.

[0225] 11. The method of embodiment 10, wherein the magnetic beads bound to the cfDNA are transferred to the elution solution by inserting a rod-shaped magnet into a washing solution containing magnetic beads bound to the cfDNA, and the magnetically bound beads are deposited in a container containing the elution solution.

[0226] 12. The method of implementation scheme 1, wherein the transfer of the magnetic beads is automatic.

[0227] 13. The method of implementation scheme 1, wherein the liquid biological sample is a plasma sample.

[0228] 14. The method of implementation scheme 1, wherein the liquid biological sample is obtained from a pregnant female.

[0229] 15. The method of implementation scheme 1, wherein the liquid biological sample is obtained from a human subject.

[0230] 16. The method of implementation scheme 1, wherein the volume of the liquid biological sample is 0.5-2 mL.

[0231] 17. The method of embodiment 1, wherein the volume of the elution solution is less than 200 µl.

[0232] 18. The method of embodiment 1, wherein the volume of the elution solution is less than or about 150 µl, less than or about 100 µl, less than or about 90 µl, less than or about 80 µl, less than or about 70 µl, less than or about 60 µl, less than or about 50 µl, or less than or about 40 µl.

[0233] 19. The method of implementation 1, wherein step (a) is carried out in a perforated plate.

[0234] 20. The method of implementation scheme 19, wherein the washing and elution steps are performed in a porous plate of the same size.

[0235] 21. The method of implementation scheme 20, wherein the perforated plate is a 24-hole deep-hole plate.

[0236] 22. The method of implementation scheme 1, wherein at least two replicate liquid biological samples containing cfDNA are contacted with a plurality of magnetic beads.

[0237] 23. The method of implementation scheme 1, wherein the magnetic beads bound to the cfDNA in each replicate sample are collected after step (a).

[0238] 24. The method of embodiment 1, further comprising, after step (a), sequentially transferring the washed cfDNA-bound magnetic beads to two or more washing solutions.

[0239] 25. The method of implementation scheme 1, wherein the magnetic beads are of uniform diameter.

[0240] 26. The method of implementation scheme 1, wherein the diameter of the magnetic beads is about 1 µm.

[0241] 27. The method of embodiment 1, wherein the washed cfDNA-bound magnetic beads are incubated in the elution solution for at least 4 minutes.

[0242] 28. The method of embodiment 1, wherein heat is applied to the elution solution containing the magnetic beads in step (d) to elute the cfDNA.

[0243] 29. The method of implementation scheme 1, wherein the magnetic plate comprises a ring magnet.

[0244] 30. The method of implementation scheme 1, wherein the magnetic plate causes the magnetic beads to adhere to the container wall in step (d).

[0245] 31. The method of embodiment 1, wherein the liquid biological sample is prepared by removing cells from a blood sample.

Claims

1. A method for separating cell-free DNA (cfDNA) from a liquid biological sample from a mammal, the method comprising: a) Directly contact the liquid biological sample containing cfDNA with multiple reversibly DNA-binding magnetic beads. b) The cfDNA-bound magnetic beads are separated from the unbound component of the liquid biological sample by inserting a rod-shaped magnet into the liquid biological sample to magnetically bind the cfDNA-bound magnetic beads, and the cfDNA-bound magnetic beads are removed from the unbound component of the liquid biological sample. c) Transferring the cfDNA-bound magnetic beads from the liquid biological sample to a container containing a washing solution; d) Transferring the cfDNA-bound magnetic beads to a container containing an elution solution by inserting a magnet into a washing solution containing the cfDNA-bound magnetic beads, and depositing the magnetically bound beads into the container containing an elution solution of less than 200 mL. µl ; and e) The magnetic beads containing the cfDNA are separated from the elution solution and the elution solution containing the cfDNA is collected by contacting a container containing the magnetic beads in the elution solution with a magnetic plate, wherein the magnetic plate comprises a ring magnet that causes the magnetic beads to adhere to the container wall, wherein the elution solution containing the cfDNA has less contamination and a higher yield compared to the elution solution in a container that has not been in contact with the magnetic plate.

2. The method of claim 1, wherein, The magnetic beads comprise cross-linked polystyrene.

3. The method of claim 1, wherein, The surface of the magnetic beads contains one or more functional groups that can reversibly bind DNA.

4. The method of claim 1, wherein, The magnetic beads are coated with a silicon-containing coating.

5. The method of claim 1, wherein, The cfDNA-bound magnetic beads are separated from the unbound components of the liquid biological sample by transferring the cfDNA-bound magnetic beads from the liquid biological sample to another container.

6. The method of claim 1, wherein, The transfer of the magnetic beads is automatic.

7. The method of claim 1, wherein, The liquid biological sample is a plasma sample.

8. The method of claim 1, wherein, The liquid biological sample was obtained from a pregnant female.

9. The method of claim 1, wherein, The liquid biological sample was obtained from human subjects.

10. The method of claim 1, wherein, The volume of the liquid biological sample is 0.5-2 mL.

11. The method of claim 1, wherein, The volume of the elution solution is less than or about 150 µl, less than or about 100 µl, less than or about 90 µl, less than or about 80 µl, less than or about 70 µl, less than or about 60 µl, less than or about 50 µl, or less than or about 40 µl.

12. The method of claim 1, wherein, Step (a) is performed in a perforated plate.

13. The method of claim 12, wherein, The washing and elution steps were performed in a multi-well plate of the same size.

14. The method of claim 13, wherein, The perforated plate is a 24-hole deep-hole plate.

15. The method of claim 1, wherein, At least two replicated liquid biological samples containing cfDNA were in contact with multiple magnetic beads.

16. The method of claim 15, wherein, After step (a), magnetic beads containing the cfDNA from each replicate sample are collected.

17. The method of claim 1, further comprising, after step (a), sequentially transferring the washed cfDNA-bound magnetic beads to two or more washing solutions.

18. The method of claim 1, wherein, The magnetic beads are of uniform diameter.

19. The method of claim 1, wherein, The diameter of the magnetic beads is approximately 1 µm.

20. The method of claim 1, wherein, The washed cfDNA-bound magnetic beads are incubated in the elution solution for at least 4 minutes.

21. The method of claim 1, wherein, Heat is applied to the elution solution containing the magnetic beads in step (d) to elute the cfDNA.

22. The method of claim 1, wherein, The liquid biological sample is prepared by removing cells from a blood sample.

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