Analysis method for storing blood for subsequent analysis of cell-free DNA

Abstract

The present invention relates to the storage and testing of anomalies of cell-free DNA (cfDNA) from blood, such as maternal blood or blood for cancer screening.

Description

Technical Field

[0001] The present invention relates to the storage and testing for abnormalities in cell-free DNA (cfDNA) from plasma, such as maternal blood or the blood of a cancer patient or a patient undergoing cancer screening. Background Art

[0002] Since its introduction into clinical care in 2011 (see Ehrich, 2011 and Palomaki, 2011), non-invasive prenatal testing (NIPT) has provided information about the risk of fetal aneuploidy to many pregnant women in different countries. Regardless of the platform used or the protocol followed, most laboratories will report a failure rate between 0 and 25%. (Badeau, 2017.) Failure is usually due to a fetal fraction that is too low to be reliable. (Blais, 2018.) Some causes of low fetal fraction are known, such as small gestational age, high BMI (body mass index), the presence of some fetal or placental abnormalities, T18, and maternal age. (Hou, 2019; Ashoor, 2013.)

[0003] Increased gestational age is associated with increased fetal fraction, but screening tests are useful early in pregnancy, and increased fetal fraction is mainly observed later in pregnancy. (Wang, 2013.) At the molecular level, fetal cfDNA can be distinguished from maternal cfDNA by the fact that it is shorter in fragments (Fan, 2010; Shi, 2020), is highly methylated, and is enriched at specific genomic locations (Wang, 2013). Recently, several studies have reported that the use of high-concentration agarose gel electrophoresis to select the shortest fragments from libraries prepared from cfDNA before sequencing can significantly improve fetal fraction (Welker, 2021; Qiao, 2019; Xue, 2020; Qiao, 2019; Hu, 2019). In the context of cancer, the same procedure also appears to improve the detection of rare mutant alleles. (Underhill, 2021.)

[0004] Tumor circulating cell-free DNA has also been shown to be shorter than cfDNA from healthy cells. (Jiang, 2015.) For both applications, different systems are used to separate or enrich these short fragments by electrophoresis using precast gels such as 2% E-gel EX from Invitrogen (Thermofisher), 2%-3% agarose cassettes from BluePippin (Sage Bioscience), and 3% agarose cassettes from Yourgene Health Canada. (Underhill, 2021.)

[0005] In order to obtain a sufficient proportion of cfDNA for meaningful sequencing, plasma must be carefully prepared to avoid contamination with host genomic DNA. Blood samples need to be processed within 4-8 hours after collection, and some people prefer to perform an additional high-speed centrifugation step to prepare plasma. (Barrett, 2011.) However, this is not always possible because the collection site is not always equipped with equipment to perform high-speed centrifugation on plasma samples before freezing the samples for shipment to a central testing laboratory. The solution adopted by most blood collection sites is to use special tubes designed to maintain the integrity of cfDNA (such as by using cross-linking agents to stabilize the cell membrane). Examples of such special tubes are Blood collection tubes. Not using specialized tubes and storing blood for long periods of time can lead to an increase in cfDNA over time due to the breakdown of genomic material. (Fernando 2018.) Specialized tubes allow blood samples to stabilize, allowing time for the sample to be transferred to a laboratory equipped to process the sample. However, these specialized tubes are expensive.

[0006] Recently, it has been reported that the use of vacutainer tubes with gel K2EDTA to collect plasma for molecular diagnostics can produce NIPT-grade cfDNA (Giroux, 2021.) The use of these tubes, followed by filtration, has been shown to be cost-effective and effective in reducing time-consuming steps in the laboratory. However, although most sites can centrifuge blood tubes, many sites are not equipped with the facilities and staff to open blood tubes and filter plasma.

[0007] Therefore, alternatives are needed for storing whole blood samples and processing blood to allow meaningful sequence analysis of cfDNA. Summary of the invention

[0008] Embodiments of the present disclosure relate to a method for analyzing nucleic acid sequences obtained from a blood sample to provide sequence information, comprising: collecting a blood sample into a blood collection device, wherein the blood collection device does not include a fixative; storing the blood at a temperature above -20°C and below 35°C (such as room temperature or refrigerated temperature) without a fixative before separating cell-free DNA from plasma; separating plasma from blood cells present in the blood sample before or after storage; separating cell-free DNA from the plasma more than 24 hours after blood collection from a subject; separating the cell-free DNA by size and separating cell-free DNA less than 300 bp; and analyzing the nucleic acid sequence of the separated cell-free DNA to detect sequence information. The blood can be stored for an extended period of time without a fixative before cfDNA is separated from the plasma. This period of time can be up to 3, 4, 5, 6, 7, 8, or 9 days after blood collection.

[0009] Suitable collection devices for use in the present invention may include a container having only one or more compositions therein, wherein the one or more compositions consist of one or more anticoagulants and metal ions selected from potassium, lithium, and sodium. An example of such a device is a K2EDTA tube. Alternatively, the blood collection device may include: a container having only one or more compositions therein, wherein the one or more compositions consist of one or more anticoagulants and metal ions selected from potassium, lithium, and sodium; and an isolation gel. In other embodiments, the collection device may include a container having only one or more compositions therein, wherein the one or more compositions contain or consist of an anticoagulant.

[0010] As mentioned above, the separation step can be used to separate cfDNA less than 300bp in the sample of the separated cfDNA. In some embodiments, the cfDNA separated in the size separation step is 185bp or less than 185bp, or 165bp or less than 165bp. In other embodiments, the cfDNA separated in the size separation step is 155bp or less than 155bp, or 150bp or less than 150bp. In another embodiment, the cfDNA portion separated from the plasma sample is 50bp or greater than 50bp, or 80bp or greater than 80bp. The separation step can be performed by any number of suitable polynucleotide size selection techniques known in the art, including gel electrophoresis, chromatography, bead-based separation methods, or membrane filters with pore sizes that hinder the passage of polynucleotides greater than the desired threshold.

[0011] After size selection, the nucleic acid sequence of the cell-free DNA separated can be sequenced and analyzed to determine the presence of genetic abnormalities in fetal DNA, or based on the presence of one or more genetic markers associated with tumorigenicity or malignant tumors, determine the sequence information about cancer cells in the subject or confirm the presence of cancer cells. In order to obtain sequence information about the cfDNA separated, it may be necessary to prepare a sequence library, and it may be necessary to amplify the cfDNA. Once size selection and library preparation are completed, the sample can be analyzed to obtain sequence information. Sequence information can be analyzed to detect mutations and chromosome abnormalities, including but not limited to translocations, inversions, monosomy, trisomy and other aneuploidies, deletions, insertions, methylations, amplifications, fragments, translocations and rearrangements or chromosome aberrations (chromathripsis). Sequence information can also be used to detect any changes in gene sequences compared to reference sequences.

[0012] Another aspect of the present disclosure is a sequence validity test that can detect abnormal performance of a size selection step or unacceptable sample degradation. In a method involving size selection of a DNA sample containing cfDNA and sequencing of the sample DNA after size selection, the method may include performing a sequence validity test on the sequence information obtained from the sequencing step. The validity test includes creating a first normalized fragment size spectrum from the sequencing information associated with the sample; comparing one or more values ​​within the fragment size spectrum with one or more corresponding value ranges obtained from a reference parameter set including multiple valid fragment size spectrums; and if one of the one or more values ​​within the fragment size spectrum associated with the sample is within the corresponding value range, accepting the sequence information and its analysis results, or if one of the one or more values ​​within the fragment size spectrum associated with the sample is outside the corresponding value range, rejecting the sequence information. The validity test may also include calculating a first relative fragment size frequency value from the sequencing information associated with the sample; comparing the first relative fragment size frequency value with a reference relative fragment size frequency value set including a plurality of valid relative fragment size frequencies; and accepting the sequence information and its analysis results if the first fragment size frequency associated with the sample is within a specified value range within the reference set, or rejecting the sequence information and its analysis results if the first fragment size frequency associated with the sample is outside the specified value range within the reference set. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Depicted is a diagram showing storage in a plasma preparation tube or Plot of fetal fractions for various samples at different time points for samples that were stored in tubes for 2-9 days.

[0014] Figure 2A Depicted is a graph showing the fetal fraction calculated from the reduction of X chromosome fragments in male pregnancy. Figure 2B Depicted is the fetal fraction assessed using the number of Y chromosome segments in males only.

[0015] Figure 3 Depicted is a graph illustrating the sizes of sequenced fragments calculated from paired-end sequencing mappings. Fragments are shown in grey before size selection and in blue after size selection.

[0016] Figure 4A study schematic for the study described in Example 3 is depicted. After recruitment, each participant donated 20 mL of blood. The blood was collected in 5 mL EDTA vacutainers and shipped to the destination laboratory for storage and processing. The blood was stored in its vacutainers at ambient temperature for 0-8 hours, 72 hours, 120 hours, or 168 hours and then processed by double centrifugation to obtain EDTA plasma. The plasma was stored at -80°C and processed at intervals once enough samples were collected for one run.

[0017] Figure 5 Depicted is a one-way ANOVA of the raw fetal fraction estimates for 268 valid samples after sequencing, which shows fetal fraction estimate data by time point, where the mean of all time points is shown by a horizontal line, green represents ANOVA, and blue represents ± standard deviation, as well as ANOVA, mean, and standard deviation analysis. Time point 1 = 0-8 hours, time point 2 = 72 hours, time point 3 = 120 hours, and time point 4 = 168 hours.

[0018] Figure 6 : Mean, standard deviation, and Welch ANOVA for 275 valid libraries in this study plus sample M056 (time point 1; 2.49 ng / μL) from run 5. Post-PCR library quantification (ng / μL) data are presented by time point. Note: Four equal variance tests indicate that variances are not equal for data at different time points; therefore, Welch ANOVA was performed instead of ANOVA. Time point 1 = 0-8 hours, time point 2 = 72 hours, time point 3 = 120 hours, and time point 4 = 168 hours. Welch ANOVA P < 0.0001 shows that there are significant differences (P < 0.05) in the library concentration data at the four time points in the study.

[0019] Fig. 7A : Size spectrum distribution and frequency of patient sample M047 at each time point, where: time point 1 = red / TP1 (YGL013-6), time point 2 = orange / TP2 (YGL015-6), time point 3 = blue / TP3 (YGL038-6), and time point 4 = green / TP4 (YGL012-6).

[0020] Figure 7B : Size spectrum distribution and frequency of patient sample M100 at each time point, where: time point 1 = red / TP1 (YGL026R-12), time point 2 = orange / TP2 (YGL003-12), time point 3 = blue / TP3 (YGL021-12), and time point 4 = green / TP4 (YGL012-12). DETAILED DESCRIPTION

[0021] A method for storing blood containing cfDNA to be sequenced is described herein. According to the present disclosure, a whole blood sample is stored in a blood collection device lacking a fixative for an extended period of more than 24 hours before cfDNA is isolated from blood or plasma to obtain sequence information. The method includes performing a size selection step on the separated DNA to select cfDNA less than a specific size, such as less than or equal to 185bp, before obtaining sequence information of the size-selected cfDNA. By selecting cfDNA with a nucleotide length less than or equal to, for example, 185bp, the fraction of fetal cfDNA in the cfDNA sample is increased, thereby improving the sample quality for meaningful sequence analysis.

[0022] 5.1. Definitions

[0023] A "blood sample" herein refers to a whole blood sample that has not been fractionated or separated into its component parts.

[0024] "Fixation" is a technique that helps maintain the structure of cells and / or subcellular components, such as organelles (e.g., nuclei), in a blood sample. Fixation changes the chemical or biological structure of cellular components, for example by cross-linking them. Fixation can hinder the lysis of whole cells and organelles, thereby inhibiting the release of cellular nucleic acids into the surrounding medium. For example, during the centrifugation of whole blood, fixation can inhibit the release of nuclear DNA from white blood cells into the plasma fraction.

[0025] A "fixative" is a reagent used to fix a blood sample.

[0026] "Target sequence" refers to a nucleic acid sequence associated with a difference in sequence representation in healthy individuals relative to diseased individuals. The target sequence can be a sequence on a chromosome that is misrepresented, i.e., over- or under-represented, in a disease or genetic condition. The target sequence can be a portion of a chromosome, i.e., a chromosome segment, or an entire chromosome. For example, the target sequence can be a chromosome that is over-represented in an aneuploid condition, or a gene encoding a tumor suppressor that is under-represented in cancer. The target sequence includes sequences that are over- or under-represented in the total population or subpopulation of cells of the subject. It can also be referred to as a "marker" of a genetic genotype associated with a specific phenotype (such as a disease).

[0027] "Sequence tag" refers to a sequence of at least 30bp read from a cfDNA strand. In some embodiments, the tag is specifically assigned (ie, mapped) to a larger sequence, such as a reference genome, by alignment.

[0028] 5.2. Methods for increasing the fraction of cfDNA and analyzing it

[0029] Embodiments of the present disclosure relate to a method for increasing the fraction of the cfDNA portion from plasma, such as from a maternal blood sample or a blood sample for cancer diagnosis, and analyzing the nucleic acid sequence of the fraction to provide sequence information. The method involves collecting a blood sample into a blood collection device. The blood collection device does not include a fixative. In some embodiments, the blood collection device consists of a container having one or more anticoagulants and metal ions selected from potassium, sodium, and lithium. In some embodiments, the blood collection device consists of a container having one or more anticoagulants and metal ions (such as potassium, sodium, or lithium) and an isolation gel.

[0030] Before or after storage, the blood sample is centrifuged in the absence of a fixative to separate plasma from blood cells. Before or after separating plasma, the blood sample can be stored at a temperature within the range of -20 ° C to 35 ° C, such as at room temperature or ambient temperature, refrigerated temperature, or a temperature between room temperature and refrigerated temperature, until the sample can be further processed. When the sample can be processed between 24-216 hours after blood collection, for example, 48 / 72 to 216 hours or 48 / 72 to 192 hours or 48 / 72 to 168 hours or 48 / 72 to 144 hours, cfDNA is separated from plasma. In order to increase the fraction of the cfDNA part, the cfDNA is separated by size, such as by gel electrophoresis, and cfDNA less than 300bp (for example, less than 185, 165, 160, 155 or 150bp) is separated. Before or after size selection, a cfDNA sample for sequencing can be prepared by optionally amplifying and generating a sequence library containing cfDNA. The size-selected and separated cfDNA can then be analyzed to detect sequence information. The steps described herein may be performed by one entity or by multiple entities.

[0031] 5.2.1 Blood collection device

[0032] The blood sample is collected in a collection device. The collection device can be a vacuum collection container, typically a tube. According to the present disclosure, the blood collection device does not contain a fixative. Examples of suitable blood collection devices include K2 or K3 EDTA blood collection tubes with or without an isolation gel.

[0033] As defined above, a fixative is a reagent used to fix a blood sample. Examples of fixatives include aldehydes (e.g., formaldehyde, glutaraldehyde, and derivatives thereof), formalin, sulfhydryl-reactive crosslinkers, carbohydrate-reactive crosslinkers, carboxyl-reactive crosslinkers, photoreactive crosslinkers, cleavable crosslinkers, AEDP (3-[(2-aminoethyl)dithio]propionic acid·HCl), APG (p-azidophenylglyoxal monohydrate), BASED, BM(PEO)3 (1,8-bis-maleimido-PEO3), BM(PEO) 4 (1,8-bis-maleimido-PEO4), BMB (1,4-bismaleimidobutane), BMDB (1,4-bis-maleimido-2,3-dihydroxybutane), BMH (bis-maleimidohexane), BMOE (bis-maleimidoethane), BS3 (bis(sulfosuccinimidyl) suberate), BSOCOES (bis(2-[succinimidyloxycarbonyloxy]ethyl)sulfone), DFDNB (1-5 DMA (dimethyl adipimide·2HCl), DMP (dimethylpimelimide·2HCl), DMS (dimethyl suberimide·2HCl), DDPPB (1,4-bis-(3'-[2'pyridyldithio]propionamido)butane), DSG (disuccinimidyl glutarate), DSP (succinimidyl disulfide propionate) (Lomant's reagent)), DSS (disuccinyl suberate) The present invention also contains sulfo-BSOCOES, sulfo-DST and sulfo-EGS.

[0034] Other fixatives may be stabilizers of cell membranes, such as DMAE (dimethylaminoethanol), cholesterol, cholesterol derivatives, high concentrations of magnesium, vitamin E and vitamin E derivatives, calcium, calcium gluconate, taurine, niacin, hydroxylamine derivatives, bimoclomol, sucrose, astaxanthin, glucose, amitriptyline, isomer A hopane tetral phenylacetate), hopane tetraphenylacetate isomer B, citicoline, inositol, vitamin B, vitamin B complex, cholesterol hemisuccinate, sorbitol, calcium, coenzyme Q, ubiquinone, vitamin K, vitamin K complex, menaquinone, zonisamide (zonegran), zinc, ginkgo extract, diphenylhydantoin, perftoran, polyvinylpyrrolidone, phosphatidylserine, tegretol, PABA, disodium cromoglycate, nedocromil sodium, phenyloin, zinc citrate, mexitil, dilantin, sodium hyaluronate, or poloxamer 188.

[0035] Other fixatives are formaldehyde-releasing preservatives such as one selected from the group consisting of diazolidinyl urea, imidazolidinyl urea, dimethoylol-5,5-dimethylhydantoin, dimethylol urea, 2-bromo-2-nitropropane-1,3-diol, oxazolidine, sodium hydroxymethylglycinate, 5-hydroxymethoxymethyl-1-1aza-3,7-dioxabicyclo[3.3.0]octane, 5-hydroxymethyl-1-1aza-3,7-dioxabicyclo[3.3.0]octane, 5-hydroxypoly[methyleneoxy]methyl-1-1aza-3,7-dioxabicyclo[3.3.0]octane, quaternaryadamantine, and any combination thereof.

[0036] In another embodiment, in addition to EDTA and / or heparin, the blood collection device may not contain agents that reduce DNA degradation, such as nuclease inhibitors. Examples of nuclease inhibitors include diethyl pyrocarbonate, ethanol, aurin tricarboxylic acid (ATA), formamide, vanadyl ribonucleoside complexes, macaloid, proteinase K, hydroxylamine-oxy-copper ions, bentonite, ammonium sulfate, dithiothreitol (DTT), β-mercaptoethanol, cysteine, dithioerythritol, tris (2-carboxyethyl) phosphine hydrochloride, and divalent cations such as Mg. +2 , Mn +2 、Zn +2 , Fe+2 , Ca +2 , Cu +2 Other agents that reduce DNA degradation may also be excluded from the device, such as zinc chloride, guanidine hydrochloride, guanidine isothiocyanate, N-lauroylsarcosine, and sodium dodecyl sulfate.

[0037] The present disclosure contemplates the exclusion of any specific combination of the above fixatives or any specific combination of the above fixatives and / or agents that reduce DNA degradation (such as nuclease inhibitors).

[0038] In some embodiments, the collection device contains an anticoagulant. The anticoagulant can be selected from ethylenediaminetetraacetic acid (EDTA), heparin, citrate, oxalate and any combination thereof. In some embodiments, the anticoagulant can be spray dried on the wall of the collection device. In other embodiments, the anticoagulant is a liquid solution in the device. Suitable solvents include water, saline, dimethyl sulfoxide, alcohol and mixtures thereof. In embodiments, the collection device includes a container having only one or more compositions, wherein the one or more compositions are composed of an anticoagulant and a metal ion (such as those selected from an alkali metal or an alkaline earth metal). In particular, the metal ion can be selected from calcium, sodium, lithium and potassium.

[0039] In embodiments with EDTA, the EDTA may be chelated with a metal ion selected from potassium, lithium, and sodium, and typically potassium. In some embodiments, the blood collection set may include a container having only one or more compositions, wherein the one or more compositions consist of chelated EDTA.

[0040] In some embodiments, the collection device contains a separation gel. Such gels are commonly used in serum separator tubes (SST) for plasma preparation (see, e.g., BD SST TM and PST TM , Becton, Dickinson, and Company). The density of the gel will be between the density of cells in the blood and the liquid phase of the blood (densities of 1.09 g / cm 3 and 1.03 g / cm 3 ). Due to its density, the isolation gel physically separates the liquid component (plasma) of the blood from the red and white blood cells after centrifugation and acts as a barrier between the liquid phase and the cells. The isolation gel is an inert gel such as a polyester gel or silica gel. Other suitable gels include sorbitol-based gels in diacrylate oligomers.

[0041] In some embodiments, a blood collection set includes: a container having only one or more compositions, wherein the one or more compositions consist of an anticoagulant and metal ions, such as chelated EDTA; and a spacer gel.

[0042] In some embodiments, the collection device contains a coagulant.

[0043] 5.2.2 Storage of blood collection devices and removal of macromolecules from plasma

[0044] In some embodiments, after collecting a blood sample from a patient, the blood can be stored at a temperature between -20°C and 35°C, such as standard ambient temperature (25°C), room temperature (20°C-24°C), refrigerated temperature (1°C-8°C) or any temperature therebetween for a period of up to 7-9 days, and then centrifuged to separate plasma from the cell fraction and extract DNA. The storage time before centrifugation can be 2, 3, 4, 5, 6, 7, 8 or 9 days. The storage temperature can be 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, or a value or range therebetween. In other embodiments, the storage temperature can be in the range of 9°C-23°C, 20°C-25°C, 25°C-30°C or 30°C-35°C. In some embodiments, the storage time does not exceed 7 days, or is between 2-5 days, 2-6 days, 2-7 days, 3-5 days, 3-6 days, 4-6 days, 3-7 days or 4-7 days. In some embodiments, the storage time is between 2-7 days or 3-7 days and is stored at refrigerated temperature, room temperature, or a temperature therebetween.

[0045] Once removed from storage (such as refrigerated storage), the sample can be fractionated to remove larger sized particles. Techniques for this are described in et al., Int. J. Mol. Sci. 2020, 21, 8634, entitled "Technical and Methodological Aspects of Cell-Free Nucleic Acids Analyzes", which is hereby incorporated by reference. In one embodiment, this is performed by centrifugation. Centrifugation can be a one-step or two-step process. In some embodiments, centrifugation is performed at a refrigerated temperature. In other embodiments, centrifugation is performed at room temperature. For the first centrifugation step, the sample is centrifuged at a low speed. Plasma is harvested from the low-speed fractionated sample. For the second step, the plasma is subjected to a low-speed or high-speed centrifugation step. Standard practice is a high-speed step. The low-speed centrifugation parameter is a parameter commonly used to separate plasma from cellular components in a blood sample. For example, for a low-speed step, the rotation speed can be between 1,000 and 2,000 x g, and the rotation time can be 8, 9, 10, 11, 12 minutes or a certain time or range therebetween. In some embodiments, the spinning speed can be 1,500-1,900 x g or 1,600-1,800 x g, and the spinning time is 9-11 minutes or 10 minutes. For the high speed step, the spinning speed can be about 3-10 times that of the low speed step in the same amount of time. For example, the high speed centrifugation can be 3,000 to 18,000 x g.

[0046] A microfiltration step can also be used instead of the second centrifugation step to remove larger particles from the plasma. In some embodiments, the microfiltration step comprises a membrane separation process to remove particles with an average molecular weight of >200 kDa, >100 kDa, >75 kDa, or >65 kDa using a membrane with a pore size of less than 3, 2, or 1 μm. In some embodiments, the pore size of the microfiltration membrane filter is 0.55 μM, 0.50 μM, or 0.45 μM, or less than 0.55 μM, 0.50 μM, or 0.45 μM.

[0047] In other embodiments, the blood sample is collected in a collection device, such as using a collection device with an isolation gel, and centrifuged at a low speed shortly after collection to separate the plasma from the cellular fraction of the blood sample. In an embodiment, this step can be performed within 6-8 hours after sample collection, and the collection device with the graded sample can be stored thereafter (i.e., plasma does not need to be separated immediately). Alternatively, the plasma can be removed from the collection device and stored in a second collection device without a fixative. The low-speed centrifugation parameters are the same as described above. The storage temperature and storage time are the same as described above. Once removed from storage, the plasma can be removed from the collection device, and if it is not removed before storage, a second centrifugation step or a filtration step can be performed according to the parameters described above.

[0048] 5.2.3 cfDNA Isolation

[0049] After collecting the plasma fraction as described above, cfDNA is separated / extracted from the plasma. Extraction is actually a multi-step process involving separation of DNA from plasma in a column or other solid-phase binding matrix. The extracted cfDNA typically includes maternal and fetal cfDNA. In some embodiments, separation can be performed by the following operations.

[0050] A. Denaturing and / or degrading proteins in the plasma (e.g., by contact with proteases) and adding guanidine hydrochloride or other chaotropic agents to the solution (to facilitate the expulsion of cfDNA from the solution).

[0051] B. Contacting the treated plasma with a support matrix (such as a membrane or beads in a column) or superparamagnetic particles with a coating to selectively bind nucleic acids. Such coatings may include silica, carboxyl, amine, imidazole, or a combination thereof.

[0052] C. Washing the support matrix.

[0053] D. Release the cfDNA from the matrix using a release solution to recover the cfDNA for downstream processes (e.g., index library preparation) and statistical analysis.

[0054] The first part of this cfDNA separation procedure involves denaturing or degrading nucleosomal proteins, and taking other measures to release DNA from nucleosomes. Typical reagent mixtures for achieving this separation include detergents, proteases, and chaotropes, such as guanine hydrochloride. Proteases are used to degrade nucleosomal proteins and background proteins in plasma, such as albumin and immunoglobulins. Chaotropes destroy the structure of macromolecules by interfering with intramolecular interactions mediated by non-covalent forces such as hydrogen bonds. Chaotropes also make components of plasma such as proteins negatively charged. Negative charge makes the medium energetically incompatible with negatively charged DNA to a certain extent. The use of chaotropes to promote DNA purification is described in Boom et al., "Rapid and Simple Method for Purification of Nucleic Acids", J. Clin. Microbiology, Vol. 28, No. 3, 1990.

[0055] After this protein degradation treatment, which at least partially releases the DNA helix from the nucleosomal proteins, the resulting solution is passed through a column or otherwise exposed to a support matrix that selectively binds the cfDNA in the treated plasma. The negative charge imparted to the media components facilitates the adsorption of the DNA into the pores of the support matrix.

[0056] After the treated plasma is passed through the support matrix, the support matrix bound to the cfDNA is washed to remove proteins and other unwanted components in the sample. After washing, the cfDNA is released from the matrix and recovered. By way of example, in the case of amine or imidazole functionalized beads, in an acidic solution, cfDNA is adsorbed to the positively charged amine or imidazole portion. In an alkaline solution, the amine and imidazole portions are negatively charged, and cfDNA is not adsorbed. Therefore, in order to remove unwanted components in the sample, the magnetic beads are fixed with a magnet and the beads are washed with an acidic washing solution. After washing, the beads are separated from the magnet, and the cfDNA can be recovered by washing with a solution of higher pH. A similar process is used for carboxyl-coated beads. In the case of carboxyl-coated beads, cfDNA reversibly binds to the beads in the presence of polyethylene glycol (PEG), depending on the salt concentration therein.

[0057] These DNA extraction processes typically lose a large portion of the available DNA in the plasma. The support matrix has a high affinity for cfDNA, which limits the amount of cfDNA that can be easily separated from the matrix. Therefore, the yield of the cfDNA extraction step can be quite low. Typically, the efficiency is much lower than 50% (for example, it has been found that the typical yield of cfDNA is 4-12 ng / ml of plasma from about 30 ng / ml of available plasma).

[0058] Commercially available kits can be used to manually and automatically isolate cfDNA, such as the QIAampDNA micro kit (Qiagen of Valencia, CA) or the NucleoSpin plasma kit (Macherey-Nagel or Duren, DE). Methods for isolating cfDNA are described in U.S. Pat. No. 10,837,055, which is hereby incorporated by reference in its entirety.

[0059] 5.2.4 Sequence library preparation

[0060] The purified cfDNA obtained from the above separation / extraction process can be used to prepare a sequencing library. In order to sequence a double-stranded DNA fragment population using a large-scale parallel sequencing system, the DNA fragment must be flanked by a known adapter sequence. The collection of such DNA fragments with adapters at either end is referred to as a sequencing library. Two examples of suitable methods for generating sequencing libraries from purified DNA are (1) attaching a known adapter to either end of the fragmented DNA in a manner based on connection (ligation), and (2) transposase-mediated adapter sequence insertion.

[0061] Methods for generating sequence libraries are described in U.S. Publication No. US2007 / 0128624, entitled “Methods for preparing libraries of template polynucleotides”, by Illumina Cambridge Ltd.; U.S. Publication No. US2013 / 0123120, entitled “Highly Multiplex PCR Methods and Compositions”, by Natera Ltd.; JF Hess et al., Library preparation for next generation sequencing: A review of automation strategies, Biotechnology Advances, Vol. 41, 2020, 107537, ISSN 0734-9750, https: / / doi.org / 10.1016 / j.biotechadv.2020.107537; and Head, Steven R et al., Library construction for next-generation sequencing: overviews and challenges, BioTechniques Vol. 56, 2 61-4, 66, 68, everywhere, Feb. 1, 2014, doi:10.2144 / 000114133, all of which are hereby incorporated by reference in their entirety. The skilled person will be aware that there are many suitable massively parallel sequencing technologies.

[0062] Sequencing libraries can be normalized using techniques known to those skilled in the art to facilitate bulk sample pooling.

[0063] 5.2.5 cfDNA Amplification

[0064] In some embodiments, the cfDNA present in the sample can be amplified before or after preparing the sequence library. Amplification can be performed by any suitable technique, such as a method based on polymerase chain reaction (PCR) or recombinase polymerase amplification (RPA). Such techniques are known to those skilled in the art. RPA is described in Lobato et al., Recombinase polymerase amplification: Basics, applications and recent advances, Trends in analytical chemistry: TRAC Vol. 98 (2018): 19-35. doi: 10.1016 / j.trac.2017.10.015, which is hereby incorporated by reference in its entirety.

[0065] In order to use the segment of PCR amplification DNA, first heat the sample to denature or divide the DNA into two sections of single-stranded DNA. Next, an enzyme called "Taq polymerase" uses the original chain as a template to synthesize and build two new DNA chains. This process leads to the replication of the original DNA, and each new molecule contains an old chain and a new chain of DNA. Then, each of these chains can be used to create two new copies, and so on. Repeat the cycle of denaturation and synthesis of new DNA. In some embodiments, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amplification cycles are carried out. More details of PCR are described in Lorenz, Todd C., Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies, Journal of Visualized Experiments, 63e3998. May 22, 2012, doi: 10.3791 / 3998, which is hereby incorporated by reference in its entirety.

[0066] 5.2.6 Size Selection

[0067] Before or after amplification, size selection is performed to increase the percentage of fetal fraction in the sample, thereby increasing its signal during sequencing. The maximum value of the required sequence length of the cfDNA separated by size selection can be 185bp, 180bp, 170bp, 175bp, 170bp, 165bp, 160bp, 155bp, 150bp or 145bp or a value therebetween. In some embodiments, the range of the required size of cfDNA is 80-160bp, 80-155bp, 80-150bp, 90-160bp, 90-155bp, 90-150bp, 100-150bp, 100-155bp, 100-160bp, 110-150bp, 110-155bp, 110-160bp, 80-170bp or 80-185bp. In some embodiments, the desired size of cfDNA ranges from 100-200 bp, 150-250 bp, 200-300 bp, and 250-300 bp.

[0068] Size selection can be performed by any technique for separating macromolecules according to size, such as gel electrophoresis, chromatography, bead-bound matrices, or membrane filtration. In the case of gel electrophoresis, the desired size range can be recovered manually or automatically using an instrument, such as Yourgene Health Canada's LightBench or NIMBUS Select.

[0069] Manual methods for recovering DNA from electrophoresis gels include: (1) cutting a gel slice corresponding to a DNA molecule band of a specific length, followed by extracting and precipitating the DNA from the gel slice; and (2) collecting one or a series of aliquots directly from the electrophoresis gel at a pipette-accessible point in the gel (referred to in the art as an "extraction well"), followed by precipitating the DNA.

[0070] An automated electrophoresis system that can be used for size selection is described in U.S. Patent No. 10,775,344 owned by Yourgene Health Canada, which is hereby incorporated by reference in its entirety. The electrophoresis system can be configured to distribute power to each gel lane, and the power can be adjusted based on monitoring current feedback on each lane. In one embodiment, the system includes an interface box having a plurality of channels, the system including: a robotic workstation for receiving at least one control signal from an external computing device to control electrophoresis operations thereon, the robotic workstation including: an arm including at least one pipette; and an on-board power module; electrically coupled to and received by a first modular base on the robotic workstation, the interface box being housed in or positioned on the base, the interface box including the plurality of channels, a pair of electrical contacts associated with each of the plurality of channels, the first modular base including a plurality of conductive cables or wires to distribute independently controllable power signals from the power module to each channel, the base including a processor and a memory, the processor being configured to: receive a power signal from the power module; receive the at least one control signal designated for the interface box of the first modular base; and modulate the power signal according to the control signal to generate an independently controllable power signal defined for each channel of the interface box of the first modular base. In one embodiment, the processor is configured to further modulate the power signal according to electrophoresis parameters stored on the memory and associated with each of the box channels. The system may also include at least a second modular base and a power transmission module electrically coupled to the power module, the power transmission module being configured to transmit a power signal as a broadcast between the power module and the first modular base and the at least second modular base. Through multiplexing, the control signal is uniquely identifiable for each of the box channels. Modulating the power signal may include modulating using at least one of: analog voltage control; pulse width modulation; duty cycle control for pulse width modulation; and frequency control for pulse width modulation. In a specific embodiment, the processor is further configured to (i.) monitor current feedback of each of the channels; and (ii.) adjust the modulated power signal of each of the channels based on the monitoring.

[0071] Other electrophoretic techniques are described as follows: capillary electrophoresis in self-coated low viscosity polymer matrices is described in Du M, Flanagan JH Jr, Lin B, Ma Y, Electrophoresis 2003 Sep;24(18):3147-53), which is hereby incorporated by reference in its entirety; selective extraction in a micro-electrophoretic device is described in Lin R, Burke DT, Burn MA, J. Chromatogr. A. 2003 Aug 29;1010(2):255-68, which is hereby incorporated by reference in its entirety; and microchip electrophoresis on low viscosity polymer matrices is described in Xu F, Jabasini M, Liu S, Baba Y, Analyst. 2003 Jun;128(6):589-92), which is hereby incorporated by reference in its entirety.

[0072] Size selection of cfDNA can also be performed by chromatography, such as chromatography on agarose or polyacrylamide gels, ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC, see Hecker KH, Green SM, Kobayashi K, J. Biochem. Biophys. Methods 2000 Nov 20;46(1-2):83-93, which is hereby incorporated by reference in its entirety), adsorption membrane chromatography (see Teeters MA, Conrardy SE, Thomas BL, Root TW, Lightfoot EN, J. Chromatogr. A. 2003 Mar 7;989(1):165-73, which is hereby incorporated by reference in its entirety); density gradient centrifugation (see Raptis L, Menard HA, J. Clin. Invest. December 1980; 66(6): 1391-9, which is hereby incorporated by reference in its entirety); and methods using nanotechnology means such as microfabrication of entropy trap arrays (see Han J, Craighead HG, Analytical Chemistry, Vol. 74, No. 2, January 15, 2002, which is hereby incorporated by reference in its entirety). Bead-based cfDNA size selection techniques that can also be used are described in Raymond et al., UltraPrep is a scalable, cost-effective, bead-based method for purifying cell-free DNA. PLoS ONE 15(6)(2020): e0231854. https: / / doi.org / 10.1371 / journal.pone.0231854, which is hereby incorporated by reference in its entirety.

[0073] If a size selection step is performed after sequence library preparation, the desired maximum length (e.g., less than 160 bp) or length range of cfDNA is the same, but in order to isolate the appropriate bands coming out of the size selection device, the target maximum or range to be isolated must be adjusted to account for adapters or other polynucleotides attached to or inserted into the cfDNA. For example, if the use of adapters increases the length of the cfDNA polynucleotide by 100 bp and the target maximum is less than 160 bp, the size selection parameter will be less than 260 bp.

[0074] By subjecting a sample from a pregnant woman to a size selection step, the percentage of fetal cfDNA in the sample to be analyzed for sequence information is increased by 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200% compared to before size selection. Figure 3 As shown in The percentage of fetal cfDNA in samples stored in the tube for the same period of time is approximately the same or greater. (SeqFF is the fetal fraction, expressed as a percentage of the total cfDNA sequenced, calculated by sequence read counts. See Kim SK et al.: Determination of fetal DNA fraction from the plasma of pregnant women using sequence read counts. Prenat Diagn 2015, 35: 810-815, which is hereby incorporated by reference in its entirety.) Similarly, when looking at male-only samples, size-selected samples have a higher percentage of cfDNA for all samples.

[0075] Similarly, by performing a size selection step on a sample for cancer diagnosis, the percentage of cfDNA from cancer cells in the sample to be analyzed for sequence information is increased by 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200% compared to before size selection.

[0076] 5.2.7 Analysis of cfDNA to obtain sequence information

[0077] Once size selection and library preparation are complete, the sample can be analyzed to obtain sequence information. The sequence information can be analyzed to detect mutations and chromosomal abnormalities, including but not limited to translocations, inversions, monosomy, trisomy and other aneuploidies, deletions, insertions, methylations, amplifications, fragments, translocations and rearrangements or chromosomal aberrations (chromathripsis). The sequence information can also be used to detect any changes in the gene sequence compared to the reference sequence.

[0078] In one embodiment, the sample is sequenced as part of a procedure for determining target sequences and for assessing copy number variation (CNV). Any of a variety of sequencing technologies can be utilized, including "first generation" sequencing, next generation sequencing, long read sequencing, or nanopore sequencing.

[0079] Some sequencing technologies are commercially available, such as the hybridization sequencing platform from Affymetrix (Sunnyvale, Calif.), the synthetic sequencing platform from Illumina (Hayward, Calif.), and nanoball-based sequencing performed by CompleteGenomics (San Jose, Calif.). Other single-molecule sequencing technologies include, but are not limited to, SMRT from Pacific Biosciences. TM Technology (see Anthony Rhoads et al., PacBio Sequencing and Its Applications, Genomics, Proteomics & Bioinformatics, Vol. 13, No. 5, 2015, pp. 278-289, ISSN 1672-0229, https: / / doi.org / 10.1016 / j.gpb.2015.08.002, which is hereby incorporated by reference in its entirety), ION TORRENT TM Technologies and, for example, nanopore sequencing developed by Oxford Nanopore Technologies (see Soni GV and Meller A. Clin Chem 53: 1996-2001

[2007] , which is hereby incorporated by reference in its entirety).

[0080] Although automated Sanger methods are considered 'first generation' technologies, Sanger sequencing (including automated Sanger sequencing) can also be used in the methods described herein. Additional suitable sequencing methods include, but are not limited to, nucleic acid imaging techniques, such as atomic force microscopy (AFM) or transmission electron microscopy (TEM).

[0081] By way of example, Illumina's sequencing technology can be used to sequence cfDNA. Illumina's technology relies on attaching cfDNA to a planar, optically transparent surface to which oligonucleotide anchors are bound. The template DNA is end-repaired to produce a 5'-phosphorylated blunt end, and the polymerase activity of the Klenow fragment is used to add a single A base to the 3' end of the blunt phosphorylated DNA fragment. This addition prepares the DNA fragment for connection to an oligonucleotide adapter, which has a single T base protrusion at the 3' end to improve the connection efficiency. The adapter oligonucleotide is complementary to the flow cell anchor. Under limiting dilution conditions, the adapter-modified single-stranded template DNA is added to the flow cell and fixed to the anchor by hybridization. The attached DNA fragments are extended and bridge-amplified to produce an ultra-high density sequencing flow cell with hundreds of millions of clusters, each containing 1,000 copies of the same template. In one embodiment, cfDNA is amplified using PCR before cluster amplification. Alternatively, a genome library without amplification is prepared, and only cluster amplification is used to enrich cfDNA (Kozarewa et al., Nature Methods6:291-295 (2009)). The template is sequenced using a robust four-color DNA synthesis sequencing technology, which uses a reversible terminator with a removable fluorescent dye. High-sensitivity fluorescence detection is achieved using laser excitation and total internal reflection optics. Short sequence reads of about 20-300bp (e.g., 150bp) can be compared with a reference genome masked by a repetitive sequence, and a specially developed data analysis pipeline software is used to identify the unique mapping of short sequence reads to the reference genome. A reference genome masked by non-repetitive sequences can also be used. Whether a reference genome masked by a repetitive sequence or a non-repetitive sequence is used, only the readings uniquely mapped to the reference genome are counted. After completing the first read, the template can be regenerated in situ to enable a second read from the other end of the fragment. Therefore, single-end or double-end sequencing of DNA fragments can be used. The DNA fragments present in the sample are partially sequenced, and the sequence tags containing the reads of a predetermined length (e.g., 36 bp) are counted and mapped to a known reference genome. In one embodiment, the reference genome sequence is the NCBI36 / hg18 sequence, which is available on the World Wide Web at genome.ucsc.edu / cgi-bin / hgGateway?org=Human&db=hg18&hgsid=166260105. Alternatively, the reference genome sequence is GRCh37 / hg19, which is available on the World Wide Web at genome.ucsc.edu / cgi-bin / hgGateway.Other public sources of sequence information include GenBank, dbEST, dbSTS, EMBL (European Molecular Biology Laboratory), and DDBJ (DNA Database of Japan). A variety of computer algorithms can be used to align sequences, including but not limited to BLAST (Altschul et al., 1990), BLITZ (MPsrch) (Sturrock and Collins, 1993), FASTA (Person and Lipman, 1988), or BOWTIE (Langmead et al., Genome Biology 10: R25.1-R25.10 (2009)), all of which are hereby incorporated by reference in their entirety.

[0082] By way of another example, 454 sequencing (Roche) can be used to obtain sequence information of nucleic acids in a test sample (e.g., cfDNA in a maternal test sample or cfDNA in a subject being screened for cancer) (e.g., as described in Margulies, M. et al. Nature 437:376-380

[2005] , which is hereby incorporated by reference in its entirety). 454 sequencing typically involves two steps. In the first step, the DNA is sheared into fragments of approximately 300-800 base pairs, and the fragments end with blunt ends. Oligonucleotide adapters are then attached to the ends of the fragments. The adapters serve as primers for fragment amplification and sequencing. For example, using adapter B containing a 5′-biotin tag, the fragments can be attached to DNA capture beads, such as streptavidin-coated beads. The fragments attached to the beads are PCR amplified in water-in-oil emulsion droplets. The result is multiple copies of cloned amplified DNA fragments on each bead. In the second step, the beads are captured in holes (picoliter-sized holes). Pyrophosphate sequencing is performed in parallel for each DNA fragment. Adding one or more nucleotides generates a light signal, which is recorded by a CCD camera in a sequencing instrument. The signal intensity is proportional to the number of nucleotides incorporated. Pyrophosphate sequencing utilizes the pyrophosphate (PPi) released when nucleotides are added. PPi is converted into ATP by ATP sulfurylase in the presence of adenosine 5' phosphosulfate. Luciferase uses ATP to convert luciferin into oxyluciferin, and this reaction produces light, which is measured and analyzed.

[0083] In another illustrative but non-limiting embodiment, sequence information can be obtained by performing ligation sequencing, which shears genomic DNA into fragments and attaches adapters to the 5' and 3' ends of the fragments to generate a fragment library. Alternatively, internal adapters can be introduced by: adapters are connected to the 5' and 3' ends of the fragments, the fragments are distributed, the distributed fragments are digested to generate internal adapters, and adapters are attached to the 5' and 3' ends of the resulting fragments to generate a paired library. Next, a cloned bead group is prepared in a microreactor containing beads, primers, templates, and PCR components. Following PCR, the template is denatured and the beads are enriched to separate the beads with the amplified template. The template on the selected beads is 3' modified to allow binding to a slide. The sequence can be determined by sequential hybridization and connecting partially random oligonucleotides with a determined central base (or base pair) identified by a specific fluorophore. After recording the color, the oligonucleotides connected are cut and removed, and the process is then repeated.

[0084] In another embodiment, the single molecule real-time (SMRT) TM ) sequencing technology obtains sequence information of nucleic acids in test samples (e.g., cfDNA in maternal test samples, cfDNA in subjects being screened for cancer). In SMRT sequencing, the continuous incorporation of dye-labeled nucleotides is imaged during DNA synthesis. A single DNA polymerase molecule is attached to the bottom surface of a single zero-mode wavelength detector (ZMW detector), and the detector obtains sequence information when phosphate-linked nucleotides are incorporated into the growing primer chain. The ZMW detector includes a restriction structure that enables the incorporation of single nucleotides to be observed by a DNA polymerase against the background of fluorescent nucleotides that diffuse rapidly in and out of the ZMW (e.g., in microseconds). It usually takes several milliseconds to incorporate nucleotides into the growing chain. During this period, the fluorescent label is excited and a fluorescent signal is generated, and the fluorescent label is cut off. The measurement of the corresponding fluorescence of the dye indicates which base is incorporated. Repeat the process to provide a sequence.

[0085] In another embodiment, sequence information of nucleic acid (e.g., cfDNA in maternal test sample or cfDNA in subject being screened for cancer) in test sample is obtained by nanopore sequencing.Nanopore sequencing DNA analysis technology is developed by multiple companies, including, for example, Oxford Nanopore Technologies (Oxford, United Kingdom), Sequenom and NABsys.Nanopore sequencing is a single molecule sequencing technology, whereby single molecule DNA is directly sequenced when it passes through nanopore.Nanopore is a small hole, and its level is usually 1 nanometer in diameter.The nanopore is immersed in a conducting fluid and an electric potential (voltage) is applied to the nanopore to generate a slight current caused by the conduction of ions through the nanopore.The amount of current flowing is sensitive to the size and shape of the nanopore.When the DNA molecule passes through the nanopore, each nucleotide on the DNA molecule blocks the nanopore to varying degrees, thereby changing the amplitude of the current passing through the nanopore to varying degrees.Therefore, this change of current provides the reading of the DNA sequence when the DNA molecule passes through the nanopore.

[0086] In another embodiment, sequence information of nucleic acids in a test sample (e.g., cfDNA in a maternal test sample or cfDNA in a subject being screened for cancer) is obtained by using a chemically sensitive field effect transistor (chemFET) assay (e.g., as described in U.S. Patent Application Publication No. 2009 / 0026082, which is hereby incorporated by reference in its entirety). In one example of this technology, a DNA molecule can be placed in a reaction chamber and the template molecule can be hybridized to a sequencing primer bound to a polymerase. The incorporation of one or more triphosphates into a new nucleic acid chain at the 3′ end of the sequencing primer can be discerned as a change in the current of the chemFET. An array can have multiple chemFET sensors. In another example, a single nucleic acid can be attached to a bead, and the nucleic acid can be amplified on the bead, and the individual beads can be transferred to separate reaction chambers on a chemFET array, each chamber having a chemFET sensor, and the nucleic acid can be sequenced.

[0087] In another embodiment, sequence information is obtained using Ion Torrent single-molecule sequencing, which pairs semiconductor technology with simple sequencing chemistry to translate chemically encoded information (A, C, G, T) directly into digital information (0, 1) on a semiconductor chip. In nature, when nucleotides are incorporated into DNA strands by polymerases, hydrogen ions are released as byproducts. Ion Torrent uses a high-density array of micromachined wells to perform this biochemical process in a massively parallel manner. Each well contains a different DNA molecule. Below the well is an ion-sensitive layer, and below that is an ion sensor. When a nucleotide, such as C, is added to the DNA template and then incorporated into the DNA strand, a hydrogen ion is released. The charge of this ion will change the pH of the solution, which can be detected by Ion Torrent's ion sensor. The sequencer - essentially the world's smallest solid-state pH meter - calls the bases, converting directly from chemical information to digital information. The Ionpersonal Genome Machine (PGM) then TM ) The sequencer sequentially injects one nucleotide after another into the chip. If the next nucleotide that floods the chip does not match, no voltage change is recorded and the base is not called. If there are two identical bases on the DNA strand, the voltage will be twice as high and the chip will record two identical bases being called. Direct detection allows nucleotide incorporation to be recorded within seconds.

[0088] In another embodiment, the method includes obtaining sequence information of nucleic acid (e.g., cfDNA in maternal test sample) in a test sample using hybrid sequencing. Hybrid sequencing includes contacting a plurality of polynucleotide sequences with a plurality of polynucleotide probes, wherein each of the plurality of polynucleotide probes can be optionally tethered to a substrate. The substrate can be a flat surface including an array of known nucleotide sequences. The hybridization pattern with the array can be used to determine the polynucleotide sequences present in the sample. In other embodiments, each probe is tethered to beads, such as magnetic beads, etc. Hybridization with beads can be determined, and used to identify a plurality of polynucleotide sequences in a sample.

[0089] In another embodiment, the method comprises obtaining sequence information of nucleic acid in a test sample (e.g., cfDNA in a maternal test sample) by massively parallel sequencing of millions of DNA fragments using Illumina's synthetic sequencing and reversible terminator-based sequencing chemistry (e.g., as described in Bentley et al., Nature 6:53-59

[2009] , which is hereby incorporated by reference in its entirety).

[0090] Mapping of sequence tags is accomplished by comparing the sequence of the tag to a reference sequence to determine the chromosomal origin of the sequenced nucleic acid molecule (cfDNA in this case) and does not require specific genetic sequence information. A small degree of mismatch (0-2 mismatches per sequence tag) can be allowed to account for slight polymorphisms that may exist between the reference genome and the genome in the mixed sample.

[0091] In one embodiment, the label of all regions (for example, all chromosomes) mapped to the reference genome is counted, and CNV, i.e., the excessive or insufficient representation of the target sequence (for example, chromosome or its part) in the mixed DNA sample is determined. The method does not need to be distinguished between two genomes.

[0092] Correctly determine whether there is CNV (for example, aneuploidy, polyploidy and deletion) required accuracy in sample based on the variation (chromosome variability) of the sequence tag quantity mapped to reference genome between sample in sequencing operation and the variation of the sequence tag quantity mapped to reference genome in different sequencing operation to predict.For example, for the label mapped to GC enrichment or GC poor reference sequence, the variation may be particularly obvious.Other variations may be caused by using different nucleic acid extraction and purification schemes, the preparation of sequencing libraries and using different sequencing platforms.Present method uses sequence dosage (chromosome dosage or fragment dosage) based on standardized sequence (standardized chromosome sequence or standardized fragment sequence) knowledge, essentially explains the cumulative variation caused by chromosome (in operation), sequencing (in operation) and platform-dependent variation.Chromosome dosage is based on the knowledge of standardized chromosome sequence, and the standardized chromosome sequence can be composed of a single chromosome, or is composed of two or more chromosomes selected from chromosome 1-22, X and Y.Alternately, standardized chromosome sequence can be composed of two or more sections of a single chromosome segment, a chromosome or two or more chromosomes. Segment doses are based on knowledge of a standardized segment sequence, which may consist of a single segment of any chromosome, or of two or more segments of any two or more of chromosomes 1-22, X, and Y.

[0093] In one embodiment, cfDNA is sequenced to determine the allele sequence of the target locus in the sample. The target heterozygous locus is identified from the sequence, and the ratio of the allele is quantified. The ratio indicates whether there is a chromosomal abnormality.

[0094] In some embodiments, the method further involves determining whether the fetus has a genetic disease based on at least one target sequence of the fetus. In some embodiments, this includes determining whether the fetus is homozygous for a pathogenic allele within the target sequence when the mother is a heterozygote for the same allele. In some embodiments, the pathogenic allele is an allele of a single nucleotide polymorphism (SNP) in the target sequence. In some embodiments, the pathogenic allele is an allele of a short tandem repeat (STR) in the target sequence.

[0095] In some embodiments, the at least one target sequence comprises a site of an allele associated with a disease. The at least one target sequence may comprise one or more of: a single nucleotide polymorphism, a tandem repeat, a microdeletion, an insertion, and an insertion-deletion (indel). In some embodiments, the method further involves determining whether the fetus is homozygous or heterozygous for the disease-associated allele.

[0096] In some embodiments, the method further relates to counting sequence tags to determine the copy number variation (CNV) of chromosome sequence, and / or checking the sequence of sequence tags to detect non-copy number variation of chromosome sequence. Some embodiments also relate to determining whether the fetus has polymorphism or chromosomal abnormality. In some embodiments, the method further relates to determining whether the fetus has SNP, tandem repeats, insertion, deletion, indel, translocation, duplication, inversion, CNV, partial aneuploidy and / or complete aneuploidy. Complete chromosome aneuploidy can be duplication, proliferation or loss of complete chromosome. In some embodiments, the method further relates to positioning partial aneuploidy. In some embodiments, the SNP is selected from single SNP and tandem SNP. In some embodiments, the tandem repeats are selected from dinucleotide repeats and trinucleotide repeats.

[0097] In some embodiments, the method further involves determining a normalized chromosome sequence value for the target chromosome sequence and comparing the sequence value to an upper threshold and a lower threshold, wherein a sequence value exceeding the upper threshold or the lower threshold determines the presence or absence of the CNV, respectively.

[0098] In some embodiments, analyzing multiple sequence tags involves determining whether there is a chromosomal abnormality in chromosomes 1-22, X, and Y. Some embodiments also involve determining whether the fetus is homozygous in an allele within the target sequence, or determining that the mother is heterozygous and the fetus is homozygous. Some embodiments also involve determining the fetal fraction of cfDNA.

[0099] Methods and systems for analyzing cfDNA are described in U.S. Pat. No. 10,837,055, which is hereby incorporated by reference in its entirety.

[0100] Another aspect of the present disclosure is a sequence validity test that can detect abnormal performance of the size selection step or unacceptable sample degradation. One embodiment of this test is described in Example 4 and is used to evaluate the suitability of the sample in Example 3. Although Example 3 describes the use of the method in the case of using a K2EDTA tube, it should be understood that this validity test can be used to evaluate the integrity of the sample subjected to the size selection step regardless of the type of collection tube or storage time or conditions. In a method involving size selection of a DNA sample containing cfDNA and DNA sequencing of the sample after size selection, the method may include performing a sequence validity test on the sequence information obtained from the sequencing step. The validity test includes: creating a first normalized fragment size spectrum from the sequencing information associated with the sample; comparing one or more values ​​within the fragment size spectrum with one or more corresponding value ranges obtained by a reference parameter set including multiple valid fragment size spectrums; and accepting the sequence information and its analysis results if one of the one or more values ​​within the fragment size spectrum associated with the sample is within the corresponding value range, or rejecting the sequence information if one of the one or more values ​​within the fragment size spectrum associated with the sample is outside the corresponding value range. The validity test may also include: calculating a first relative fragment size frequency value from the sequencing information associated with the sample; comparing the first relative fragment size frequency value with a reference relative fragment size frequency value set including a plurality of valid relative fragment size frequencies; and accepting the sequence information and its analysis results if the first fragment size frequency associated with the sample is within a specified value range within the reference set, or rejecting the sequence information and its analysis results if the first fragment size frequency associated with the sample is outside the specified value range within the reference set.

[0101] Example

[0102] 5.3. Example 1

[0103] This study is a comparative study that evaluated the quality of cfDNA extracted from blood stored and transported in K2EDTA tubes with gel (after low centrifugation) and uncentrifuged Streck tubes. Previous work has shown that EDTA-gel tubes are as good as Streck tubes in collecting blood and preparing cfDNA for NIPT without reducing fetal fraction if the blood is processed on the same day (Giroux, 2021). This study tested the feasibility of keeping centrifuge tubes at 4°C for five days and shipping them cold to a sequencing laboratory 4800 km away.

[0104] Pregnant women were recruited only in Vancouver after they agreed to participate in the study. The maternal age of all recruited pregnant women was between 10 and 14 weeks. The details of the study population are shown in Table 1.

[0105] Table 1.

[0106]

[0107]

[0108] For each participant, two 10 ml peripheral blood samples were collected in Streck tubes, catalog number 218997 (Streck, NB, USA), and 8 ml were collected in K2EDTA gel blood tubes for molecular diagnosis, catalog number GR-455040 (Greiner, Austria). Within 6 hours after sample collection (as recommended by the manufacturer), the EDTA-gel tubes were centrifuged at 1800 g for 10 min and then kept at 4 ° C until cold transported to the laboratory in Quebec City once a week. The blood collected in the Streck tubes was transported to the same laboratory at room temperature twice a week. A total of 61 samples were collected in Vancouver and transported across 4800 km to the testing laboratory in Quebec City.

[0109] Plasma was carefully collected from the Streck tube using a 1 ml pipette 2 to 5 days after blood collection. The plasma was filtered through a 0.45 μM HPF-Millex-PVDF-Durapore from Millipore (Merck, MA, USA) using a 5 ml syringe. The plasma in the K2EDTA tube was decanted into a 5 ml syringe barrel and filtered using the same type of filter as previously reported (Giroux, 2021). The EDTA-Gel tube took 2 to 9 days between blood collection and filtration.

[0110] cfDNA was prepared from 2 to 5 ml of plasma using the QIAamp circulating nucleic acid kit catalog number 55114 (Qiagen, Germany). 50 or 70 μl of DNA was recovered and quantified by fluorescence using the Qubit dsDNA HS assay kit catalog number Q32851 (Thermofisher, MA, USA). According to the manufacturer's instructions and with some modifications, the KAPA Hyper Prep kit catalog number 07962363001 (Roche, Switzerland) was used to prepare the library using a volume of 15 to 25 μl containing 2 to 15 ng of cfDNA. The volume of each step was reduced by half. Kapa Unique double transposition adapter catalog number 08861919702 (Roche, Switzerland) was used with a final concentration of 0.5 μM. 10 amplification cycles were performed and post-amplification cleanup was performed with 0.6X-1X Kapa beads catalog number 07983298001 (Roche, Switzerland).

[0111] For one set of samples, the library was quantified by fluorescence using the Qubit dsDNA HS assay kit as described above. The molar concentration was calculated using an average size of 325 base pairs (bp). Up to 17 pools of equimolar libraries were prepared and double-end sequencing was performed for 150 cycles on an Illumina Next-Seq 550 instrument with a medium output flow cell catalog number 20024909 (Illumina, CA, USA).

[0112] For the second set of samples, the libraries were size selected using LightBench from Yourgene Health Canada (Manchester, UK). 25 μl of sample plus loading buffer and 200 bp / 300 bp labeled markers were run together on a 3% precast gel. The software was programmed to extract DNA in the range of 229 bp to 286 bp. The recovered DNA was quantified with Qubit and the mass was converted to molar concentration using an average size of 275 bp. Pools of up to 25 libraries were prepared and sequenced on a medium output flow cell as described above.

[0113] result

[0114] Using standard procedures with Streck tubes, samples arrived at room temperature within 5 days. Seven women had a fetal fraction less than 4%, and six of these had a weight greater than 70 kg or a BMI greater than 25.

[0115] Using EDTA-gel tubes, samples also arrived within 5 days, but must be kept refrigerated. Most samples arrived within two days through the priority shipping (Priority Shipping) of the selected transporter. However, two shipments involving 14 samples were delayed by 4 and 5 days, and these samples were no longer kept refrigerated. These 14 samples were affected during transportation, and the fetal score was much lower than expected. Their average fetal score was reduced by 4.6% compared with the average fetal score obtained using Streck tubes. The EDTA-gel tubes (N=47) that arrived refrigerated in two days or less were similar to the fetal scores observed with Streck tubes, but were reduced by an average of 1.1%. There is a trend between the time spent at 4°C (keeping the tubes in the refrigerator until transportation) and the degree of fetal score reduction. We observed that the fetal score decreased from 0.75% in 2-3 days to 1.41% in 6 days or more with the increase of time before treatment.

[0116] Using LightBench from Yourgene Health Canada TM Instrument, all libraries prepared from DNA isolated from plasma from EDTA-gel tubes were run on 3% gel electrophoresis. The software was set to acquire fragments of 229 base pairs to 286 base pairs, which takes into account the addition of 136 bp on the cfDNA strand by the adapter. The acquired material was sequenced and fetal fraction assessed with SeqFF as usual. An increase in fetal fraction was observed in all but five samples. Those five samples that did not show an increase initially had higher fetal fractions. The samples with a five-day shipping delay were those that had less of an increase compared to the Streck tube results. Two samples had fetal fractions that were 0.4% and 3% smaller, respectively, compared to the Streck tube, but the fetal fraction was improved compared to the EDTA-gel tube before size selection, and an additional twelve samples had increases of 0.1% to 1.5% ( Figure 1 , left sample). The remaining 47 samples all increased by an average of 5.4%, ranging from 0.8% to 10.2%, with the exception of 3 samples that remained above 10%, but decreased compared to Streck or even EDTA-gel before size selection ( Figure 1 , samples with stars). Determining fetal fraction using SeqFF gives a rough estimate, but is still very useful for female pregnancies. Fetal fraction calculated from the reduction in ChrX fragments in male pregnancies was also determined and found to have a significant increase in fetal fraction for each sample analyzed ( Figure 2A ). Even samples with low SeqFF fetal scores will appear significantly higher when calculated using this method ( Figure 1 and Figure 2AThe tubes used and selection on 3% agarose did not artificially increase the fetal fraction by interfering with SeqFF counts, as an increase in Y chromosome reads was also observed in pregnancies with male fetuses (n=25). (Supplement Figure 2B ).

[0117] Figure 3 The sizes of the sequenced fragments calculated from the mapping of paired-end sequencing are shown. The fragments are gray before size selection and blue after size selection.

[0118] Table 2

[0119] Streck EDTA - All EDTA -4℃ EDTA +SS Average FF(%) 8.6 6.8 7.6 12.6 FF range (%) 2.3-26.8 0.4-25.2 0.4-25.2 6.4-22.6 Number of samples with w / FF < 4% 7 16 9 0

[0120] Example 2

[0121] The aim of the second study is to evaluate the effect of extended storage time prior to centrifugation of whole blood collected in K2EDTA tubes on the detection of fetal cfDNA. Individual tubes from a single blood draw from each participant will be incubated at a series of time points, centrifuged to obtain plasma, and then processed to isolate and amplify DNA, perform size selection, generate DNA libraries, and sequence cfDNA. Fetal fraction percentage and chromosome ratios will be used to determine the effect of storage time prior to centrifugation. This will enable measurement of the effect of variable blood storage time on fetal fraction percentage.

[0122] Samples will be processed at the following time points: Time Point 1–0-8 hours, Time Point 2–72 hours, Time Point 3–120 hours, and Time Point 4–168 hours. During this period, a portion of the samples will be stored at refrigerated temperature and a portion will be stored at room temperature. At the appropriate time points, plasma will be separated from whole blood via centrifugation. All tubes will be centrifuged at 1600g x 10min within 6h of sample collection. Plasma from the EDTA tube will be poured into a syringe barrel and filtered through a 0.45μm Millipore filter. The separated plasma samples will be stored at -80°C until at least 96 samples have been collected, at which time DNA extraction will begin. Storage at this temperature will preserve the samples until extraction. Extraction of cfDNA can be performed using the QIAamp Circulating Nucleic Acid Kit Catalog No. 55114 (Qiagen, Germany). Yourgene Health Canada Will be used to select cfDNA of a specific size in the sample. Size selection parameters are the same as in Example 1. NGS sequencing libraries are prepared and sequenced on the Illumina system. Fetal fraction is estimated using SeqFF.

[0123] Example 3

[0124] The aim of the third study was also to evaluate the effect of extending the storage time of whole blood in K2EDTA blood tubes (also known as K2EDTA vacuum blood collection tubes) before centrifugation on fetal cfDNA testing. Individual tubes of a single blood draw from each participant were incubated at time points 1 - 4, centrifuged twice to obtain plasma, and then processed to isolate and amplify DNA, generate a DNA sequencing library, pool the sequencing libraries, perform size selection and sequence the cfDNA. Fetal fraction percentage and chromosome ratios were used to determine the effect of storage time before centrifugation. This enabled the measurement of the effect of variable blood storage times on fetal fraction percentage and the quality of NIPT testing in standard blood tubes with delayed plasma separation.

[0125] Samples were stored at ambient temperature and processed at the following time points: time point 1 – 0 - 8 hours, time point 2 – 72 hours, time point 3 – 120 hours, and time point 4 – 168 hours. The ambient temperature range was 20 °C to 35 °C. At the appropriate time points, plasma was separated from whole blood via double centrifugation. The plasma separation centrifugation process included a first centrifugation step at 1600 rpm for 10 minutes and a second centrifugation step at 3000 rpm for 10 minutes. After the first centrifugation, the plasma from the K2EDTA tube was transferred to a second tube and the second centrifugation step was performed. After the second step, plasma was removed from the second tube, transferred to a third tube and stored at -80 °C until enough samples had been collected to start a < / = 48 sample run, at which point DNA extraction was started. The separated plasma samples were stored at -80 °C until approximately 48 samples had been collected, at which point the DNA extraction process was started. Samples were stored at this temperature until extraction.

[0126] cfDNA was extracted using the MagMAX TM Cell-Free DNA Isolation Kit. Although Streck tubes were not used, the lysis step was performed as part of the protocol and had no adverse effect on samples collected in EDTA tubes. QS250 from Yourgene Health Canada TM was used to select cfDNA of a specific size in the samples. The size selection parameters were 106 - 146 bp (values excluding adapter length), and the recovery range was approximately 100 - 186 bp (average fragment length of approximately 143 - 146 bp). NGS sequencing libraries were prepared and sequenced on an Illumina system. SeqFF was used to estimate the fetal fraction.

[0127] Sample set details

[0128] A set of 105 matched clinical samples was collected for this study. Samples were collected with known gestational age, height, weight, and BMI. Individual vacutainers from a single blood draw from each participant were incubated for time points 1-4, centrifuged to obtain plasma, and then run Nx Workflow. Fetal fraction percentage and chromosome ratios were used to determine the effect of storage time prior to centrifugation.

[0129] From almost all participants, 4-5 mL blood samples (called matched samples) were collected from each of 105 patient volunteers (pregnant women >10+0 weeks of gestation) in a BD Vacutainer TM Plastic K2EDTA tubes. For each participant, after incubation at ambient temperature, matched 5 mL aliquots were centrifuged at each time point 1 to 4. Table 3 below provides a summary of the samples collected. Since the samples obtained from the participants were less than 20 mL, the time points that deviated from 105 samples were less than 105 samples.

[0130] Table 3

[0131]

[0132] Participants were designated M029 to M105. The following samples were mistakenly subjected to a single centrifugation instead of the standard double centrifugation in the IONA Nx protocol:

[0133] M029-M30 Time Points 1-3

[0134] M031-M032 time points 1-2.

[0135] Due to the use of a single spin method for preparing plasma, these samples have been excluded from this report.

[0136] The remaining samples were double centrifuged and are summarized as follows:

[0137] M029-M030 Time Point 4

[0138] M031-M032 Time Points 3 and 4

[0139] M033-M105 time points 1, 2, 3 and 4.

[0140] The above samples are those included in the analysis.

[0141] Participant exclusion and inclusion criteria

[0142] The exclusion and inclusion criteria were as follows:

[0143] Inclusion criteria

[0144] Pregnant women ≥ 18 years old

[0145] Women who can understand and provide informed consent

[0146] Single-baby pregnancy ≥ 10+0 weeks, no upper limit on gestational period, i.e. until the due date

[0147] Exclusion criteria

[0148] Known maternal aneuploidy or maternal cancer

[0149] Twin or missing twin cases

[0150] Known infectious diseases, such as current COVID-19 infection or during COVID-19 quarantine, hepatitis B

[0151] Anyone considered to be a vulnerable group

[0152] Anyone known to have had an organ or stem cell transplant

[0153] Anyone known to have had a blood transfusion in the past 12 months

[0154] result

[0155] Sample Summary

[0156] A summary of the QC validity status of all double-spun patient samples at the four time points used in this study is shown below in Table 4.

[0157] Table 4

[0158]

[0159]

[0160]

[0161]

[0162] Table 1: Summary of all patient samples, time points, and sample validity status for all double-spin samples used in this study, including 1x duplicate sample validity. LP = Library Preparation; * = M056 (time point 1) was pooled and size selected in run 5, although invalid as described in Section 8.4; Sample unavailable = sample not received for processing; Omitted = single spin sample; Omitted for other reasons.

[0163] Library validity

[0164] The library preparation (:LP) efficiency ratio was calculated for each run and each corresponding time point, where post-PCR quantification values ​​≥ 2.5 ng / μL were considered valid.

[0165] The overall initial library effectiveness and ratio effectiveness ratios after 1x re-testing of all samples at each time point in the study are shown in the following table. Figure 5 Shown in.

[0166]

[0167] Table 5: Initial library efficacy and 1x retest ratios at each time point and overall efficacy ratios as part of this study. Time point 1 = 0-8 hours, time point 2 = 72 hours, time point 3 = 120 hours, and time point 4 = 168 hours

[0168] Summary statistics (mean and standard deviation) and Welch ANOVA tests were performed on the 275 valid libraries after 1x retest (see Table 5) plus sample M056 (time point 1) from run 5 that was included in the sample pool for size selection because it nearly met the library validity criterion (2.49 ng / μL). Data for all 276 samples are in Figure 6 Shown in.

[0169] The Welch ANOVA test was performed instead of ANOVA because the unequal variance test indicated that the standard deviations of the data at each time point were unequal (see Figure 6 ); therefore, ANOVA is not an appropriate analysis method because this test assumes equal variances.

[0170] It can be observed that the data show that the library concentration increases with the storage time of the samples in the K2EDTA tubes before centrifugation. Time point 4 shows the maximum average library concentration (11.31 ng / μL, Figure 6 Error! Reference source not found. ), while time point 1 showed the lowest mean concentration (6.12 ng / μL, Figure 6 Error! Reference source not found. ). Time point 2 presented the largest standard deviation (SD = 2.90, Figure 6 ), time point 4 presented the smallest standard deviation (SD = 1.48, Figure 6 Error! Reference source not found. ). Time point 1, time point 2, and time point 3 have standard deviations of 2.48, 2.90, and 2.35, respectively, indicating comparable variance between these time points. These observations are consistent with the hypothesis that extended storage of samples in EDTA tubes results in increased DNA input into the library preparation reactions; therefore, higher library concentrations would be expected to return at later time points.

[0171] The Welch ANOVA p value < 0.0001 in Table 6 indicated that there were statistically significant differences in the library quantification data between the various time points.

[0172] It should be noted that, in theory, any larger DNA fragments (>1 kb) in the starting sample could be retained based on size alone during the sample cleanup step in the library preparation workflow, regardless of whether adapter ligation / amplification was successful. Therefore, this material may contribute to the fluorescent signal in the library quantitation assay, thereby facilitating increased library concentration at later time points and extended sample storage time in studies ( Figure 6 ).

[0173] The library concentration data of the above 276 data points were processed by main effects analysis using the variability index data quality assessment in JMP to estimate which portion of the variability in the library concentration data was caused by the study variable (EDTA storage time) and the runs performed. The variance component results from this test are shown in Table 6 below.

[0174] Table 6.

[0175]

[0176] Table 6 Variance components analysis of library concentration data by run number and time point for the 276 samples analyzed in the study.

[0177] Variance component analysis showed that the study variable – EDTA storage time (time point) – contributed 50% of the variance seen in the library concentration data (Table 6). Therefore, 50% of the variance observed in the library concentration data came from other workflow variables; 10% of this variance was attributed to run-to-run variability, and the remaining 40% was attributed to variables not specifically investigated in this analysis / study (Table 6). Possible variables may include, but are not limited to, multiple operators performing sample centrifugation and plasma separation at different time points, laboratory / workflow variability of runs 1-9 (e.g., service lab runs 1-3 versus R&D runs 4-9), and variability associated with the patients from whom the samples were collected.

[0178] Sequencing effectiveness

[0179] In order to assess the quality of the DNA samples and exclude any samples with sequencing process problems that affect the quality of the sequence results, each sequencing sample was analyzed for several parameters, mainly run control and sequencing quality control for the entire run, fragment counts, fragment size profiles (compared to what is expected for cfDNA size-selected sequencing libraries), GC profiles, fetal fractions, and consistency checks. All samples must have a fetal fraction of at least ≥ 2%. In addition, all samples with a risk of false negative or false positive results are evaluated using our proprietary dynamic fetal fraction assessment. This adapts the desired fetal fraction level of the sample to support the quality and quantity of the sequencing data.

[0180] The overall sequencing validity ratio for all samples tested (11 samples were retested once) was 272 / 274 (99.27%). Eleven (11) samples that initially failed sample validity were retested in subsequent runs; nine (9) of these samples returned valid results when retested.

[0181] The ratios of initial and 1x repeat sequencing efficiencies for each time point are presented below in Table 7. Error! Reference source not found.

[0182] Table 7

[0183]

[0184] Table 7: Initial sequencing efficiency and 1x retest ratios for each time point and overall efficiency ratios as part of this study. Time point 1 = 0-8 hours, time point 2 = 72 hours, time point 3 = 120 hours, and time point 4 = 168 hours.

[0185] After 1x retesting, a total of eight (8) samples failed the sequencing validity check.

[0186] Of the six (6) samples that failed sequencing and were not retested:

[0187] 2x sequencing failure was due to fragment size profile failure at position 160 bp (M080, time point 4 and M085, time point 4)

[0188] 4x sequencing failures were due to failure of the fragment size profile at the 180 bp position (M089, time point 4; M101, time point 3; M101, time point 4 and M102, time point 4).

[0189] Of the two (2) samples that failed sequencing validity after 1x retest:

[0190] 1x sequencing failure is due to fragment size spectrum failure at position 180bp (M068, time point 3)

[0191] The failure of 1x sequencing was due to the failure of the fragment size profile at two positions, 120 bp and 180 bp (M076, time point 3).

[0192] The number of samples failing sequencing QC checks (particularly the "abnormal fragment size profile" check) increased at later time points, likely due to increased DNA input due to prolonged sample storage in EDTA tubes, resulting in heteroduplex formation by the PCR platform and library fragments in later PCR cycles. Therefore, sequencing heteroduplex library fragments after enrichment of samples by size selection (on QS250) is expected to increase the risk of samples failing IONA software QC checks; in fact, time point 4 had the highest incidence of samples failing after initial sequencing validation and 1x retest (Table), most commonly "abnormal fragment size profile". However, repeating samples by library preparation and sequencing was able to produce valid results.

[0193] An example of changes in fragment size frequency with increasing storage time prior to centrifugation can be observed in

[0194] For the two patients who returned valid results at each time point.

[0195] Fig. 7A and 7B The representative shift in fragment profiles to the right (i.e., larger DNA library insert sizes) for patient samples M047 and M100 at all four time points shown in is an expected result of heteroduplex formation; this itself is a result of increased DNA input into the library preparation due to prolonged sample storage in EDTA tubes. It should be noted that Fig. 7A and 7B All aligned DNA fragments represented by the spectra in the Figure 3 were within the expected size range for mononucleosomal cfDNA units, rather than the expected DNA fragments generated by cell lysis caused by prolonged storage time before centrifugation and plasma separation.

[0196] Fetal fraction percentage

[0197] The "IONA FF" method applies one of two algorithms to produce estimates, depending on the value measured by the chromosome count analysis. IONAFF looks at the ratio of X and Y chromosomes. Then

[0198] If the confidence that the sample is a male fetus is very high, the "E1" estimation algorithm is used. This estimates the FF from the X and Y chromosome data using a linear model that has been calibrated during development.

[0199] Otherwise (i.e., it is not certain that it is a male fetus), the "E2" algorithm is used for the sample. This method makes estimates based on a model that relates the relative frequencies of long and short fragments to the FF. This is also a linear model, but each run is calibrated using the already calculated E1 (X / Y based) estimates. This improves the accuracy of the size-based estimates compared to using a static model by eliminating run-to-run variations in the measurements.

[0200] This estimate is then output to the rest of the system together with an internal estimate of the measurement uncertainty which is also used by the validity checker.Another way to calculate the fetal fraction is to use an algorithm known in the art as SeqFF.

[0201] After analysis by IONA software, the fetal fraction percentage was determined based on the data of all 268 valid samples. A one-way ANOVA was also performed on the raw fetal fraction data (the enriched fetal fraction estimate obtained from the raw sequencing data) to assess the comparison between each time point. ANOVA of all fetal fraction data was performed in Figure 5 Shown in.

[0202] It can be observed that the data show a decreasing trend in percent fetal fraction with increasing storage time prior to centrifugation. When analyzing all 268 fetal fraction estimates, all time points presented similar standard deviations, which was supported by the unequal variance test mentioned above, indicating that there was comparable variance between these time points.

[0203] Figure 5 ANOVA p value < 0.0001 indicated that there were significant differences in the percentage of fetal fraction between the time points.

[0204] Nonetheless, 267 of 268 samples had a fetal fraction greater than 4%, a common threshold for NIPT testing, and all samples passed the IONA Nx FF cutoff.

[0205] Analysis of rare autosomal aneuploidies

[0206] All 272 (100%) valid samples returned a valid RAA plugin result of "Not Detected", indicating that no aneuploidy was detected.

[0207] Consistency analysis

[0208] The concordance analysis for each time point of valid samples in this study was performed only in cases where a valid baseline result (time point 1) was returned, even in cases where there was no result at another corresponding time point (i.e., due to library or sequencing failure), in which case an invalid result refers to a "not calculated" result relative to the SCA / FSD determination. Concordance analysis was also performed on patient samples that returned valid results at all four time points.

[0209] RAA consistency analysis

[0210] The concordance analysis of RAA status relative to baseline (Time Point 1) is summarized below in Table 11. Samples were considered where a valid Time Point 1 RAA result was available concurrently with at least one other time point.

[0211] Table 11

[0212]

[0213] Table 11. Concordance analysis of RAA status relative to efficacy baseline (time point 1) results.

[0214] Patient samples from time points 2-60 / 60 (100%) returned "not detected" RAA results consistent with baseline.

[0215] Patient samples from time points 3–62 / 62 (100%) returned “not detected” RAA results consistent with baseline.

[0216] Patient samples from time points 4–59 / 59 (100%) returned “not detected” RAA results consistent with baseline.

[0217] The consistency analysis of RAA status relative to baseline for the fifty-five (55) patient samples that returned valid results at all four time points is summarized in Table 12 below.

[0218] Table 12

[0219]

[0220] Table 12. Concordance analysis of RAA status relative to valid baseline results for fifty-five (55) patient samples that returned valid results at all time points.

[0221] Samples from time point 2 - 55 / 55 (100%) returned "not detected" RAA results consistent with baseline.

[0222] Samples from time points 3-55 / 55 (100%) returned "not detected" RAA results consistent with baseline.

[0223] Samples from time points 4–55 / 55 (100%) returned “not detected” RAA results consistent with baseline.

[0224] T13 / 18 / 21 consistency analysis

[0225] The consistency analysis of aneuploidy status at T13 / 18 / 21 relative to baseline (Time Point 1) is summarized in the table below.

[0226] Table 13

[0227]

[0228]

[0229] Table 13. Consistency analysis of T13 / 18 / 21 status results relative to effective baseline (time point 1).

[0230] Patient samples from time point 2-60 / 60 (100%) returned "low risk" results consistent with baseline.

[0231] Patient samples from time points 3–62 / 62 (100%) returned “low risk” results consistent with baseline.

[0232] Patient samples from time points 4–59 / 59 (100%) returned “low risk” results consistent with baseline.

[0233] The consistency analysis of T13 / 18 / 21 aneuploidy status relative to baseline for fifty-five (55) patient samples that returned valid results at all four time points is summarized in the following table.

[0234] Table 14

[0235]

[0236] Table 14. Concordance analysis of T13 / 18 / 21 status relative to valid baseline results for fifty-five (55) patient samples that returned valid results at all time points.

[0237] Samples from time point 2 - 55 / 55 (100%) returned "low risk" results consistent with baseline.

[0238] Samples from time points 3–55 / 55 (100%) returned “low risk” results consistent with baseline.

[0239] Samples from time points 4–55 / 55 (100%) returned “low risk” results consistent with baseline.

[0240] Example 4

[0241] This example provides an implementation of parameters for a sequence validity check that looks at the fragment size spectrum and determines whether the spectrum is normal or abnormal. The purpose of this check is to reduce the risk of performance degradation in NIPT tests due to sample degradation and / or reduced effectiveness of the fetal DNA size selection step (also known as fetal DNA enrichment) by detecting insufficient enrichment and invalidating the corresponding sample analysis so that it does not produce a test result. The test can also be used to test cancer cell sequences in cell-free DNA.

[0242] Fetal cell-free DNA is known to exist in a specific size range that is different from maternal cell-free DNA. Therefore, by calculating a normalized fragment size profile from sequencing data associated with a sample and comparing its parameters to a pre-calculated reference parameter set that can distinguish between efficient enrichment and inefficient enrichment and / or excessive sample degradation, instances of inefficient enrichment and / or excessive sample degradation can be detected.

[0243] Size spectrum validity checks are designed to, for example, ensure that:

[0244] 1. Enrichment of the fetal cell-free DNA component from the maternal cell-free DNA is effective; the enrichment may be performed by size selection as described herein by ensuring preferential selection of fragments in a size range that is more likely to be fetal, and / or

[0245] 2. The sample sequencing data had a DNA fragment size profile consistent with the biological characteristics of cell-free DNA material extracted from plasma drawn from pregnant women.

[0246] One or more size envelopes are defined at specific target fragment size values. (In Example 3, the target size fragment size values ​​are 120 bp and 180 bp fragments.) In essence, each envelope encapsulates a fragment size spectrum at these points that are considered valid. Therefore, if the incoming fragment size spectrum meets the condition of being enveloped by at least one of the limits defined in this way, the size spectrum validity check of the corresponding sample is considered to have passed.

[0247] Configuration parameters: To configure this implementation of the validity check parameters, the following parameters are required. They should be stored in an efficient hierarchical representation (e.g., using an XML-based format).

[0248] The number of defined size limits – N 极限 (integer, mandatory), 108.

[0249] Array of size limits – size limits [·]. For each entry:

[0250] O size number of checkpoints – N 检查点(integer, mandatory).

[0251] O arrays of size checkpoints - size checkpoints[·]. For each entry:

[0252] Defined fragment size – S 检查点 (integer, mandatory).

[0253] Valid relative frequency range – lower limit – z L (real number, optional).

[0254] Notice :If for z U If a value is provided, this value may be omitted, in which case the lower limit is considered to be zero. \

[0255] Valid relative frequency range – upper limit – z U (real number, optional).

[0256] Notice :If for z L This value may be omitted if one is provided, in which case the upper limit is considered to be one.

[0257] Test input data: The input data is an array of fragment size values ​​measured in the sequencing data.

[0258] Test Output: The method produces a single output, as follows: Size Spectrum Validity Status – V SP (boolean, mandatory).

[0259] Calculation of sample size profiles: First, a relative frequency distribution is formed from the array of fragment size measurements generated for the sample, as generated by the analysis core pipeline via methods appropriate for the sequencing platform used. This distribution will be calculated as an array consisting of the frequency of each size occurring, normalized by the total number of fragments included in the distribution, thus:

[0260]

[0261] Where n s is the number of fragments of size s present for all fragment sizes ∈ S, and S is the set of all fragment sizes measured by the sequencing data, where contributing fragments are considered to be only those with sizes 13, 18, 21, X, and Y. outside It should be noted that where N fragments are found to align at the same genomic position, and thus deduplication occurs, each fragment of size s will have a fragment count of n. s Contribution 1 / N.

[0262] For any size value that is not represented (i.e., ), the corresponding relative frequency distribution value Z[s] is considered to be zero.

[0263] Calculation of the sample validity status: The relative frequency distribution Z calculated for the sample is compared with each limit defined in the configuration parameter set in turn. For each limit, this comparison is performed as follows:

[0264] 1. At each size checkpoint, use the corresponding size value S 检查点 Find the relative frequency value of this size in the calculated distribution of the sample, that is: Z[S 检查点 ].

[0265] 2. Compare the distribution value thus obtained with the effective frequency range parameter Z L and z U Compare. If it No Falling in the interval [z L , z U ), that is: Z[S 检查点 ]<Z L or Z[S 检查点 ]≥Z U , the checkpoint comparison is marked as failed. If the distribution value is within the interval [z L , z U ), the comparison is marked as passed.

[0266] The comparison continues to the next size checkpoint within the current limit until no checkpoints remain.

[0267] Then, the limit comparison result is calculated as the logical "and" of all component checkpoint results. That is, the limit comparison is considered to have passed only when the limit comparison passes all component checkpoints.

[0268] If any single limit comparison is found to have passed, then a valid result is generated for the sample validity via the size spectrum validity check, and it is reported as V SP =True. Only one limit comparison needs to succeed for the validity check as a whole to give a valid result.

[0269] If all composition checkpoints are found to have failed after all limit comparisons are completed, an invalid result is generated for the sample validity via the size spectrum validity check, and it is V SP = False. This corresponds to an abnormal fragment size profile and the sample does not pass the sequence validity check and is rejected. A new blood draw is required.

[0270] Equivalent

[0271] Although the present invention has been described in detail with reference to its specific embodiments, it will be appreciated that functionally equivalent variations are within the scope of the present invention. In fact, various modifications of the present invention will become apparent to those skilled in the art from the aforementioned description and accompanying drawings, except those shown and described herein. Such modifications are intended to fall within the scope of the appended claims. Those skilled in the art will recognize or will be able to determine many equivalents of the specific embodiments of the invention described herein without using excessive routine experiments. Such equivalents are intended to be covered by the following claims.

[0272] All publications mentioned in this specification are incorporated by reference into the specification herein to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety herein.

[0273] As will be apparent to those skilled in the art, many modifications and variations may be made thereto without departing from the spirit and scope of the present invention. The particular embodiments described herein are provided by way of example only, and the present invention is limited only by the scope of the appended claims and equivalents to which such claims are entitled.

Claims

1. A method for analyzing a nucleic acid sequence obtained from a blood sample to provide sequence information, the method comprising the following steps: a. analyzing the nucleic acid sequence of the size-selected cell-free DNA to detect sequence information, wherein the size-selected cell-free DNA is obtained from a blood sample, wherein the blood sample is collected in a blood collection device that does not contain a fixative, wherein the blood sample is stored at a temperature greater than -20°C and less than 35°C without a fixative prior to separating the cell-free DNA from the plasma, wherein the plasma is separated from the blood cells present in the blood sample before or after storage, wherein the cell-free DNA is isolated from the plasma at least 48 hours after the blood sample is collected from the subject, and The size-selected cell-free DNA analyzed is obtained by separating the extracted cell-free DNA by size and isolating the cell-free DNA smaller than 300 bp from the extracted cell-free DNA.

2. A method for analyzing a nucleic acid sequence obtained from a blood sample to provide sequence information, the method comprising the following steps: a. analyzing the nucleic acid sequence of the size-selected cell-free DNA to detect sequence information, wherein the size-selected cell-free DNA is obtained from a blood sample, wherein the blood sample is collected in a blood collection device comprising a container having therein only one or more compositions, wherein the one or more compositions consist of one or more anticoagulants and a metal ion selected from the group consisting of potassium, lithium and sodium, wherein the blood sample is stored in the blood collection device at a temperature greater than -20°C and less than 35°C before separating the cell-free DNA from the plasma, wherein the plasma is separated from the blood cells present in the blood sample before or after storage, wherein the cell-free DNA is isolated from the plasma at least 48 hours after the blood sample is collected from the subject, and The size-selected cell-free DNA analyzed is obtained by separating the extracted cell-free DNA by size and isolating the cell-free DNA smaller than 300 bp from the extracted cell-free DNA.

3. A method for analyzing nucleic acid sequences obtained from a blood sample to provide sequence information, the method comprising the following steps: a. analyzing the nucleic acid sequence of the size-selected cell-free DNA to detect sequence information, wherein the size-selected cell-free DNA is obtained from a blood sample, The blood sample is collected in a blood collection device, which includes: a container having only one or more compositions therein, wherein the one or more compositions consist of one or more anticoagulants and a metal ion selected from the group consisting of potassium, lithium and sodium; and a barrier gel; wherein the blood sample is stored in the blood collection device at a temperature greater than -20°C and less than 35°C before separating the cell-free DNA from the plasma, wherein the plasma is separated from the blood cells present in the blood sample before or after storage, wherein the cell-free DNA is isolated from the plasma at least 48 hours after the blood sample is collected from the subject, and The size-selected cell-free DNA analyzed is obtained by separating the extracted cell-free DNA by size and isolating the cell-free DNA smaller than 300 bp from the extracted cell-free DNA.

4. The method of claim 1 or 3, wherein the cfDNA is isolated from the plasma at least 3, 4, 5, 6, 7 or 8 days after blood collection.

5. The method of any of the preceding claims, wherein the plasma is separated from the blood after storage for at least 2, 3, 4, 5, 6, 7 or 8 days after blood collection.

6. The method of claim 5, wherein the plasma is separated from the blood after storage for at least 2, 3, 4, 5 or 6 days, or after storage for 3 to 6 days, or after 3, 4 or 5 days after blood collection.

7. The method of any of the preceding claims, wherein the blood sample is stored in the blood collection device at a temperature greater than 0°C and less than 35°C or less than 30°C or less than 25°C or less than 20°C prior to separating the cell-free DNA from the plasma.

8. The method of any of the preceding claims, wherein the blood sample is stored in the blood collection device at a temperature greater than 0°C and less than 35°C or less than 30°C or less than 25°C or less than 20°C prior to separating the plasma from the blood.

9. The method of any of the preceding claims, wherein the blood sample is stored in the blood collection device at a temperature greater than 0°C or greater than 2°C and less than 15°C or less than 10°C or less than 8°C or less than 6°C prior to separating the cell-free DNA from the plasma.

10. The method of any of the preceding claims, wherein the blood sample is stored in the blood collection device at a temperature greater than 0°C or greater than 2°C and less than 15°C or less than 10°C or less than 8°C or less than 6°C prior to separating the plasma from the blood.

11. The method of any of the preceding claims, wherein the blood sample is stored at ambient temperature in the blood collection device prior to separation of the plasma from the blood.

12. The method of any of the preceding claims, wherein the nucleic acid sequence of the isolated cell-free DNA is analyzed to determine the presence of a genetic abnormality in the fetal DNA.

13. The method of any one of the preceding claims, wherein the sequence information comprises a sequence of a plurality of sequence tags, and the method further comprises: The sequences of the plurality of sequence tags are analyzed to determine the presence of at least one target sequence in the fetal DNA, wherein at least a portion of the plurality of sequence tags map to the at least one target sequence.

14. A method as claimed in any one of the preceding claims, wherein the sequence information comprises genetic mutations in genes, Optionally, wherein the gene is selected from the group consisting of BRCA1, BRCA2, MSH6, MSH2, MLH1, RET, PTEN, ATM, H-RAS, p53, ELAC2, CDH1, APC, AR, PMS2, MLH3, CYP1A1, GSTP1, GSTM1, AXIN2, CYP19, MET, NAT1, CDKN2A, NQ01, trc8, RAD51, PMS1, TGFBR2, VHL, MC4R, POMC, NROB2, UCP2, PCSK1, PPARG, ADRB2, UCP3, glur1, cart, S ORBS1, LEP, LEPR, SIM1, TNF, IL-6, IL-1, IL-2, IL-3, IL1A, TAP2, THPO, THRB, NBS1, RBM15, LIE, MPL, RUNX1, Her-2, glucocorticoid receptor, estrogen receptor, thyroid receptor, p21, p27, K-RAS, N-RAS, retinoblastoma protein, Wiskott-Aldrich (WAS) gene, factor V Leiden, factor II (prothrombin), methylenetetrahydrofolate reductase, cystic fibrosis, LDL receptor, HDL receptor, superoxide dismutase gene, and SHOX gene; or The gene is selected from the group consisting of a gene involved in nitric oxide regulation, a gene involved in cell cycle regulation, a tumor suppressor gene, an oncogene, and a gene associated with neurodegeneration.

15. The method of claim 7, wherein the genetic mutation is a marker for a cancer type.

16. The method of any one of the preceding claims, wherein the separation step is performed using gel electrophoresis.

17. The method of any of the preceding claims, wherein the portion of the cfDNA from the plasma sample is less than 185 bp or less than 180 bp or less than 165 bp.

18. The method of any of the preceding claims, wherein the portion of the cfDNA from the plasma sample is less than 155 bp or less than 150 bp.

19. The method of claim 17 or 18, wherein the portion of the cfDNA from the plasma sample is greater than 50 bp or greater than 80 bp or greater than 90 bp or greater than 100 bp.

20. The method of claim 1, wherein the fixative is a cross-linking agent, a metabolic inhibitor, or a membrane stabilizer.

21. The method of any one of the preceding claims, wherein the collection means comprises EDTA and a metal ion selected from potassium.

22. The method of claim 21, wherein the collection device further comprises an isolation gel.

23. The method of any one of the preceding claims, wherein the sample is obtained from a human source.

24. The method of any one of the preceding claims, wherein the subject is a pregnant female or a patient in need of cancer diagnosis.

25. The method of any of the preceding claims, wherein a sequence library comprising the cell-free DNA is generated before or after the cfDNA is isolated and size fractionated.

26. The method of claim 18, wherein the cell-free DNA is amplified before or after forming the sequence library.

27. The method of any one of the preceding claims, wherein the separation step is performed using a bead-based binding matrix, column chromatography or a membrane filter.

28. The method of any of the preceding claims, wherein the blood collection device is stored at a temperature no greater than 8°C prior to extracting the cell-free DNA from the plasma.

29. The method of any of the preceding claims, wherein the blood collection device is stored at a temperature no greater than 25°C prior to extracting the cell-free DNA from the plasma.

30. The method of any one of the preceding claims, wherein the blood collection device is stored at a temperature no greater than 30°C prior to extracting the cell-free DNA from the plasma.

31. The method of any of the preceding claims, wherein the blood collection device is stored at a temperature greater than 0°C prior to extracting the cell-free DNA from the plasma.

32. The method of any of the preceding claims, wherein analyzing the nucleic acid sequence of the isolated cell-free DNA to detect sequence information comprises sequencing the isolated cell-free DNA or sequencing at least a portion of the cell-free DNA to determine the nucleic acid sequence of one or more polynucleotides in the isolated cell-free DNA.

33. The method of any of the preceding claims, wherein the sequence information comprises a nucleic acid sequence having an insertion, a deletion, a chromosomal aberration or a methylation compared to a reference sequence.

34. The method of any of the preceding claims, wherein the nucleic acid sequence of the isolated cell-free DNA is analyzed for abnormal chromosome number or for the presence of XX or XY chromosomes by calculating chromosome ratios.

35. A method as described in any of the preceding claims, creating a first normalized fragment size spectrum from sequencing information associated with the sample; comparing one or more values ​​within the fragment size spectrum with one or more corresponding value ranges obtained by a reference parameter set including multiple valid fragment size spectrums; and accepting the sequence information and its analysis results if one of the one or more values ​​within the fragment size spectrum associated with the sample is within the corresponding value range, or rejecting the sequence information if one (or alternatively, all) of the one or more values ​​within the fragment size spectrum associated with the sample is outside the corresponding value range.

36. The method of claim 34, wherein the first normalized fragment size profile comprises or consists of: Fragment size of less than 185 bp or less than 180 bp or less than 165 bp or less than 155 bp or less than 150 bp.

37. The method of claim 34 or 35, wherein the first normalized fragment size spectrum comprises or consists of fragment sizes greater than 50 bp, or greater than 80 bp, or greater than 90 bp, or greater than 100 bp.

38. The method of any one of claims 34 to 36, wherein in the event that the sequence information of a sample is rejected, a request is submitted to redraw blood from the patient.

39. A method as described in any of the preceding claims, calculating a first relative fragment size frequency value from sequencing information associated with the sample; comparing the first relative fragment size frequency value with a reference relative fragment size frequency set including a plurality of valid relative fragment size frequencies; and accepting the sequence information and its analysis results if the first fragment size frequency associated with the sample is within a specified value range within the reference set, or rejecting the sequence information and its analysis results if the first fragment size frequency associated with the sample is outside the specified value range within the reference set.

40. The method of claim 38, wherein the first relative fragment size frequency value is a fragment size of less than 185 bp or less than 180 bp or less than 165 bp or less than 155 bp or less than 150 bp.

41. The method of claim 38 or 39, wherein the first relative fragment size frequency value is a fragment size greater than 50 bp or greater than 80 bp or greater than 90 bp or greater than 100 bp.

42. The method of any one of claims 38 to 40, wherein in the event that the sequence information of a sample is rejected, a request is submitted to redraw blood from the patient.

43. A method as described in any one of claims 38 to 41, comprising performing the calculation and comparison steps as described in claim 38 for a second relative fragment size frequency value having a bp value different from the first fragment size frequency, wherein if all (or one) of the second fragment size frequency values ​​associated with the sample are within a specified value range within the reference set, the sequence information and its analysis are accepted, or if all (or one, two or three) of the second fragment size frequencies associated with the sample are outside the specified value range within the reference set, the sequence information is rejected.

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