Systems and methods are provided herein for preparing a non-naturally-occurring preparation of DNA from perfusion fluid or flush, the preparation being useful for assessing transplantation outcomes of a donor organ or donor tissue which was perfused with the perfusion fluid or prepared for transplantation with flush. The preparation may also be useful for evaluating quality of the organ or tissue. Analysis of the DNA may be used to make clinical decisions. Analysis of the DNA may also be used for adaptive control of a mechanical perfusion system used for preserving the organ or tissue, by generating and executing appropriate parameter adjustments based on characteristics of the sampled DNA.
C12Q 1/6883 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
2.
NON-INVASIVE METHODS OF ASSESSING TRANSPLANT REJECTION IN PREGNANT TRANSPLANT RECIPIENTS
The present disclosure provides methods for preparation and analysis of biological samples of maternal transplant recipients, wherein the methods comprise extracting cell-free DNA from the recipient, wherein the cell-free DNA comprises donor-derived cell-free DNA, recipient-derived cell-free DNA, and fetal-derived cell-free DNA, and measuring amounts of cell-free DNA and donor-derived cell-free DNA enables assessment of transplant rejection. The detection of the donor-derived cell-free DNA may be performed based on loci at which the maternal transplant recipient and biological father of the fetus are homozygous and the transplant donor is heterozygous.
C12Q 1/6883 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
G16B 20/20 - Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
3.
METHODS FOR DETERMINATION AND MONITORING OF XENOTRANSPLANT REJECTION BY MEASURING NUCLEIC ACIDS OR PROTEINS DERIVED FROM THE XENOTRANSPLANT
The present disclosure provides methods for preparation and analysis of biological samples of xenotransplant recipients, wherein the methods comprise extracting fragmented or intact cell-free DNA, RNA (such as mRNA or miRNA), or protein derived from sample of the xenotransplant recipient, and measuring amounts of cell-free DNA, RNA (such as mRNA or miRNA), or protein derived from the xenotransplant enables assessment of xenotransplant rejection. The detection of the cell-free DNA, or RNA may be performed by preparing sequencing libraries and performing whole genome sequencing.
C12Q 1/6883 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
Provided herein are compositions to be used as a positive control for detection of one or more microdeletions of interest in a sample. The positive control can be used to determine an error and an efficiency rate for assays used to identify microdeletions such as 22ql 1.2 deletion (DiGeorge syndrome), chromosome 5pl5.2 (Cri-du-chat), lp36 deletion, 15ql l.2~ql3 deletion (Prader-Willi syndrome), and/or 15ql l~ql3 (Angelman syndrome).
C12Q 1/6827 - Hybridisation assays for detection of mutation or polymorphism
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
The present disclosure provides methods for preparation and analysis of biological samples of transplant recipients or subjects suffering from a disease or disorder, wherein the methods comprise extracting fragmented or intact RNA (such as mRNA or miRNA) derived from sample of the transplant recipient or subject suffering from a disease or disorder, wherein the extracted RNA comprises target RNA molecules preselected to enable assessment of transplant rejection. The detection of the target RNA molecules and total amount of RNA derived from the donor organ can be used to determine and/or monitoring transplant rejection.
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/6883 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
6.
PREDICTIVE MACHINE LEARNING MODELS FOR PREECLAMPSIA USING ARTIFICIAL NEURAL NETWORKS
Disclosed is an approach that may include generating and/or using a predictive machine learning classifier comprising one or more artificial neural networks. The classifier is configured to output a prediction related to developing preeclampsia (e.g., early onset preterm preeclampsia) during a current pregnancy of a patient based on health characteristics and one or more DNA metrics. The health characteristics and DNA metrics, such as total cell-free DNA (cfDNA) and fetal fraction (FF), may be obtained during a routine and non-invasive or minimally-invasive prenatal screening. The predictive machine learning classifier may be generated by applying deep learning techniques to data on subjects in a cohort. The data may comprise features corresponding to outcomes of prior pregnancies, health indicators, and one or more cfDNA measurements. First trimester risk assessment for preterm preeclampsia can identify patients most likely to benefit from preventative treatment protocols with a minimal or low level of intervention.
G16H 50/30 - ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for individual health risk assessment
The invention provides methods for preparing a preparation of amplified DNA derived from a biological sample of a patient who has been diagnosed with cancer useful for determining relapse or metastasis of cancer, comprising (a) sequencing DNA isolated from hematopoiesis cells in a blood or bone marrow sample of the patient or a fraction thereof to determine the presence or absence of one or more clonal hematopoiesis of indeterminate potential (CHIP) mutations; (b) sequencing (i) DNA isolated from a tumor biopsy sample of the patient or (ii) cell-free DNA isolated from the blood or bone marrow sample or a fraction thereof, to identify a plurality of patient-specific somatic mutations associated with the cancer; (c) preparing a preparation of amplified DNA by performing targeted multiplex amplification on cell-free DNA isolated from a longitudinally collected biological sample of the patient or a fraction thereof to amply a plurality of target loci to obtain amplified DNA, wherein each of the target loci spans a patient-specific somatic mutation identified in step (b) and does not span any CHIP mutation identified in step (a), wherein the biological sample is a blood, urine, or bone marrow sample; and (d) analyzing the preparation of amplified DNA by sequencing the amplified DNA to determine the presence or absence of the patient-specific somatic mutations, wherein the presence of two or more patient-specific somatic mutations associated with the cancer and the presence of one or more CHIP mutations are indicative of relapse or metastasis of the cancer.
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
The present disclosure provides methods for preparing a preparation of amplified DNA derived from a blood sample of a pregnant woman useful for identifying pregnancies having high risks of preterm birth, preeclampsia, small for gestational age, spontaneous termination, and/or non-livebirth, comprising: (a) extracting cell-free DNA from the blood sample; (b) performing targeted multiplex amplification on the extracted DNA to amplify 200-20,000 SNP loci in a single reaction volume; and (c) performing high-throughput sequencing on the amplified DNA to obtain sequence reads and using the sequence reads to determine the ploidy state of the one or more chromosomes of interest; wherein a fetal fraction of less than 2.8% and/or no-call of the ploidy state of the one or more chromosomes of interest is indicative of pregnancies having high risks of preterm birth, preeclampsia, small for gestational age, spontaneous termination, and/or non-livebirth.
C12Q 1/6883 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
The invention provides methods for preparing a preparation of amplified DNA derived from a biological sample of a pregnant woman useful for identifying neoplasm in a pregnant woman, comprising: (a) isolating cell-free DNA from a biological sample of a pregnant woman comprising a mixture of fetal cell-free DNA and maternal cell-free DNA; (b) preparing a preparation of amplified DNA by performing targeted multiplex amplification on the isolated cell-free DNA to amplify at least 100 polymorphic loci; (c) analyzing the preparation of amplified DNA by sequencing the amplified DNA to obtain sequence reads of the at least 100 polymorphic loci and using the sequence reads to identify copy number variations (CNVs) in fetal and maternal chromosomes or chromosomal segments of interest, and identifying neoplasm in the pregnant woman by the presence of two or more of CNVs in the maternal chromosomes or chromosomal segments of interest.
C12Q 1/6883 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
The invention provides methods for determining the growth rate of ctDNA, comprising (a) sequencing nucleic acids isolated from a biological sample of a cancer patient to identify patient-specific cancer mutations; (b) quantify the amount of ctDNA in a first liquid biopsy sample collected from the cancer patient by performing a multiplex amplification reaction to amplify target loci from cfDNA isolated from the first liquid biopsy sample, wherein each target locus spans at least one patient-specific cancer mutation, and sequencing the amplified target loci to identify the patient-specific cancer mutations and quantify the amount of ctDNA in the first liquid biopsy sample; (c) quantify the amount of ctDNA in a second liquid biopsy sample collected from the cancer patient by performing a multiplex amplification reaction to amplify target loci from cfDNA isolated from the second liquid biopsy sample, wherein each target locus spans at least one patient-specific cancer mutation, and sequencing the amplified target loci to identify the patient-specific cancer mutations and quantify the amount of ctDNA in the second liquid biopsy sample; and (d) determining the growth rate of the ctDNA between the first and second liquid biopsy samples.
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
Torque teno virusTorque teno virus (TTV) in a blood, plasma, serum, or urine sample of a transplant recipient; (b) measuring the amount of donor-derived cell-free DNA in a blood, plasma, serum, or urine sample of the transplant recipient; and (c) determining whether the amount of donor-derived cell-free DNA or a function thereof exceeds a cutoff threshold indicating transplant rejection and whether the transplant recipient has an increased or decreased amount of TTV indicating decreased or increased immune response, respectively.
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/6883 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
C12Q 1/70 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
12.
METHODS FOR DETECTION OF DONOR-DERIVED CELL-FREE DNA IN TRANSPLANT RECIPIENTS OF MULTIPLE ORGANS
The present disclosure provides methods of amplifying and sequencing DNA, comprising: extracting cell-free DNA from a blood, plasma, serum or urine sample of a transplant recipient who has received transplantation of one or more organs including simultaneous or sequential transplantation of multiple organs, wherein the extracted cell-free DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA; performing targeted amplification at 200-50,000 target loci in a single reaction volume using 200-50,000 primer pairs, wherein the target loci comprise polymorphic loci and non-polymorphic loci; sequencing the amplification products by high-throughput sequencing to obtain a sequencing reads and quantifying the amount of donor-derived cell-free DNA and the amount of total cell-free DNA based on the sequencing reads; and determining whether the amount of donor-derived cell-free DNA or a function thereof exceeds a cutoff threshold indicating transplant rejection or graft injury.
C12Q 1/6883 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
The present disclosure provides methods for quantifying the amount of total cell-free DNA in a biological sample, comprising: isolating cell-free DNA from the biological sample, wherein a first Tracer DNA composition is added before or after isolation of the cell-free DNA; performing targeted amplification at 100 or more different target loci in a single reaction volume using 100 or more different primer pairs; sequencing the amplification products by high-throughput sequencing to generate sequencing reads; and quantifying the amount of total cell-free DNA using sequencing reads derived from the first Tracer DNA composition.
C12Q 1/6883 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
14.
METHODS FOR ASSESSING GRAFT SUITABILITY FOR TRANSPLANTATION
This invention relates to methods and compositions for assessing the suitability of a graft for transplantation by measuring total and/or specific cell-free nucleic acids (such as cf-DNA) and/or cell lysis. Specifically, the method comprising obtaining an amount of total short fragment cf-DNA and/or graft-specific short fragment cf-DNA released from a potential graft (e.g., ex vivo), e.g., prior to contacting of the potential graft with blood cells of a potential recipient, and/or subsequent to contacting of the potential graft or cells thereof with blood cells from a potential recipient, and assessing the amount(s) to determine the suitability of the potential graft for transplantation.
The disclosure herein provides methods and compositions for detecting or monitoring immune cell populations in biological samples. The methods and compositions disclosed herein are particularly useful for detecting or monitoring immune cell populations in patients suffering from a disease or undergoing treatment of a disease resulting in depletion of immune cells. In particular, the present disclosure provides method for using multiplex PCR combined with next-generation DNA sequencing to detect DNA containing recombined V(D)J gene segments which can be used to detect immune cells.
C12Q 1/6881 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
Provided herein are improved methods of determining the sequences of cell-free DNA (cfDNA). The methods in certain embodiments are used for the analysis of circulating DNA in serum samples, such as circulating fetal DNA, circulating donor derived DNA, or circulating tumor DNA. In certain embodiments, the methods include selectively enriching trinucleosomal, dinucleosomal, mononucleosomal or sub-mononucleosomal DNA from the isolated cfDNA.
The invention provides methods for characterizing and analyzing circulating cells. In particular, the invention provides methods for confirming the identity of an individual cell and that the obtained sample was derived from a single cell of a defined identity. Additional methods are provided for analyzing single cell samples to determine copy number variation and aneuploidy in the context of circulating fetal cells, microdeletions, single nucleic acid variations associated with cancer, or early relapse of cancer and metastasis.
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
18.
METHODS AND SYSTEMS FOR CALLING PLOIDY STATES USING A NEURAL NETWORK
A method of calling a ploidy state using a neural network includes determining, for a training sample, genetic sequencing data or genetic array data for a plurality of genetic positions, determining respective true ploidy state values for a plurality of genetic segments, each genetic segment respectively comprising at least some of the plurality of genetic positions, based on the genetic sequencing data or genetic array data, and determining a neural network comprising one or more layers for calling respective ploidy state values, the neural network defined at least in part by a plurality of weights. The method further includes iteratively modifying the weights using specific processes. The method further includes calling, for a test sample, a ploidy state for a target genetic region by propagating genetic sequencing data for the test sample or genetic array data for the test sample through the modified neural network.
The present disclosure provides methods for determining the status of an allograft within a transplant recipient from genotypic data measured from a mixed sample of DNA comprising DNA from both the transplant recipient and from the donor. The mixed sample of DNA may be preferentially enriched at a plurality of polymorphic loci in a way that minimizes the allelic bias, for example using massively multiplexed targeted PCR.
C12Q 1/6883 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
A method for calling a mutation includes determining, for each target base of a plurality of target bases, a respective value for a background error parameter based on training data. The method further includes determining a motif- specific error model including the background error parameter by performing processes that include: identifying a respective motif for each target base of the plurality of target bases, grouping the plurality of target bases into a plurality of groups, each group corresponding to a particular motif, and determining, for each group, a respective motif-specific parameter value for the background error parameter based on the determined values for the background error parameter for the target bases included in each group. The method further includes calling a mutation using the motif-specific error model and sequencing information for a biological sample.
The invention provides methods for detecting single nucleotide variants in breast cancer, bladder cancer, or colorectal cancer. Additional methods and compositions, such as reaction mixtures and solid supports comprising clonal populations of nucleic acids, are provided. For example, provided here is a method for monitoring and detection of early relapse or metastasis of breast cancer, bladder cancer, or colorectal cancer, comprising generating a set of amplicons by performing a multiplex amplification reaction on nucleic acids isolated from a sample of blood or urine or a fraction thereof from a patient who has been treated for a breast cancer, bladder cancer, or colorectal cancer, wherein each amplicon of the set of amplicons spans at least one single nucleotide variant locus of a set of patient-specific single nucleotide variant loci associated with the breast cancer, bladder cancer, or colorectal cancer; and determining the sequence of at least a segment of each amplicon of the set of amplicons that comprises a patient-specific single nucleotide variant locus, wherein detection of one or more patient-specific single nucleotide variants is indicative of early relapse or metastasis of breast cancer, bladder cancer, or colorectal cancer.
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
22.
METHODS FOR ISOLATING NUCLEIC ACIDS WITH SIZE SELECTION
Disclosed here is a method for isolating nucleic acids from a biological sample, comprising: (a) contacting a first composition comprising nucleic acids obtained from a biological sample with a first matrix under a low-stringency binding condition having less than 1% aliphatic alcohols that binds less than 5% of nucleic acids of shorter than about 118 bp and more than 30% of nucleic acids longer than about 194 bp to a first matrix; and (b) contacting a second composition comprising remainder of the first composition with a second matrix under a high-stringency binding condition having less than 1% aliphatic alcohol that binds more than 70% of nucleic acids longer than about 72 bp and 30% of nucleic acids longer than about 50 bp to the second matrix. Further disclosed is a kit for isolating nucleic acids from a biological sample, comprising (a) a first binding buffer for establishing a low-stringency binding condition having less than 1% aliphatic alcohols that binds less than 5% of nucleic acids shorter than about 118 bp and more than 30% of nucleic acids longer than about 194 bp to a matrix, and (b) a second binding buffer for establishing a high-stringency binding condition having less than 1% aliphatic alcohol that binds more than 70% of nucleic acids longer than about 72 bp and 30% of nucleic acids longer than about 50 bp to the matrix.
Disclosed here is a composition comprising a primer that is (a) a loopable primer comprising a target-specific section, an adaptor section, and a stem-forming section, wherein the stem-forming section is hybridizable to a portion of the target-specific section to form a stem structure, or (b) a split primer comprising a first target-specific section, a second target-specific section, and an adaptor section positioned between the first target-specific section and the second target-specific section, or (c) a split-loopable primer comprising a first target-specific section, a second target-specific section, a stem-forming section positioned between the first target-specific section and the second target-specific section, and an adaptor section, or comprising a first adaptor section, a second adaptor section, a stem-forming section positioned between the first adaptor section and the second adaptor section, and a target-specific section. Also disclosed is a method for amplifying a target locus of interest from a template DNA, comprising at least two pre-amplification cycles using the loopable primer, the split primer and/or the split-loopable primer, wherein each amplification cycle comprises annealing the primer to the template DNA or pre-amplification product thereof and elongating the annealed primer. Further disclosed is a kit for amplifying a target locus of interest, comprising the loopable primer, the split primer, and/or the split-loopable primer.
Disclosed here is a composition for isolating nucleic acids from a biological sample, comprising a chaotropic compound and a solvent, wherein the solvent comprises a nitrile compound, tetrahydrofuran, or a combination thereof. Also disclosed is a method for binding nucleic acids to a matrix, comprising: contacting the nucleic acids from a biological sample with the matrix in the presence of a chaotropic compound and a solvent, wherein the solvent comprises a nitrile compound, tetrahydrofuran, or a combination thereof, thereby binding the nucleic acids to the matrix. Further disclosed is a kit for isolating nucleic acids from a biological sample comprising a binding buffer, wherein the binding buffer comprises a chaotropic compound and a solvent, wherein the solvent comprises a nitrile compound, tetrahydrofuran, or a combination thereof.
The present disclosure provides methods and compositions for sequencing nucleic acid molecules and identifying individual sample nucleic acid molecules using Molecular Index Tags (MITs). Furthermore, reaction mixtures, kits, and adapter libraries are provided.
The invention relates to subject-specific methods for detecting recurrence of tumours based on an understanding of the clonal/subclonal mutation profile of the subject's tumour and detection of the mutations in their cell-free DNA (cfDNA), typically by multiplex PCR of tumour mutations such as single nucleotide variants (SNVs).
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
27.
METHODS FOR CHARACTERIZING COPY NUMBER VARIATION USING PROXIMITY-LITIGATION SEQUENCING
Disclosed here is a method for detecting genome rearrangement in a biological sample, comprising: obtaining a contact matrix plotted from proximity ligation sequencing data of at least one chromosome; identifying an abnormal contact pattern in the contact matrix compared to the contact matrix of a reference genome; comparing the abnormal contact pattern in the contact matrix to one or more known patterns associated with genomic rearrangement to identify a type of genomic rearrangement causing the abnormal contact pattern. Also disclosed is a method for detecting genome rearrangement in a biological sample, comprising: selecting linked chromosomal fragments from proximity ligation sequencing data of at least one chromosome, identifying an abnormal covalent bonding pattern of the linked chromosomal fragments compared to a reference genome; and comparing the abnormal covalent bonding pattern to one or more known patterns associated with genomic rearrangement to identify genomic rearrangement causing the abnormal covalent bonding pattern.
The invention provides methods and compositions for detecting a mutation in a target gene in a sample of blood or a fraction thereof, including in certain examples, a fraction that includes circulating tumor DNA. The methods can include a tiling PCR reaction, for example a one-sided multiplex tiling reaction. Virtually any type of mutation can be detected with the methods and compositions. In certain embodiments, gene fusions are detected. Improved PCR methods, especially for performing nested multiplex PCR reactions are provided.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
G06F 19/18 - for functional genomics or proteomics, e.g. genotype-phenotype associations, linkage disequilibrium, population genetics, binding site identification, mutagenesis, genotyping or genome annotation, protein-protein interactions or protein-nucleic acid interactions
The invention provides methods for detecting single nucleotide variants in lung cancer, especially stage 3a lung adenocarcinoma and lung squamous cell carcinoma. Additional methods and compositions, such as reaction mixtures and solid supports comprising clonal populations of nucleic acids, are provided.
The invention provides improved methods, compositions, and kits for detecting ploidy of chromosome regions, e.g. for detecting cancer or a chromosomal abnormality in a gestating fetus. The methods can utilize a set of more than 200 SNPs that are found within haploblocks and can include analyzing a series of target chromosomal regions related to cancer or a chromosomal abnormality in a gestating fetus. Finally the method may use knowledge about chromosome crossover locations or a best fit algorithm for the analysis. The compositions may comprise more than 200 primers located within haplotype blocks known to show CNV.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
G06F 19/16 - for molecular structure, e.g. structure alignment, structural or functional relations, protein folding, domain topologies, drug targeting using structure data, involving two-dimensional or three-dimensional structures
31.
DETECTING MUTATIONS AND PLOIDY IN CHROMOSOMAL SEGMENTS
The invention provides methods, systems, and computer readable medium for detecting ploidy of chromosome segments or entire chromosomes based on phasing of the alleles and determination of individual and joint probabilities and a best-fit model. In some aspects, the invention provides methods, systems, and computer readable medium for detecting cancer or a chromosomal abnormality in a gestating fetus. The invention also provides methods for detecting circulating tumor nucleic acids based on the level of allelic imbalance present at the polymorphic loci found by ploidy determination. The invention also provides a method for detecting single nucleotide variants based on an estimation of amplification efficiency and error rate and a method for detecting single nucleotide variants based on a median variant allele frequency for a plurality of control samples from individuals.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
G06F 19/16 - for molecular structure, e.g. structure alignment, structural or functional relations, protein folding, domain topologies, drug targeting using structure data, involving two-dimensional or three-dimensional structures
Embodiments of the invention include methods and compositions for producing proficiency testing standards for noninvasive prenatal genetic diagnostics and for the detection and monitoring of cancer. The compositions can comprise a plurality of different nucleosomal DNA fragments derived from either primary cells or cell lines. The amount of the different nucleosomal DNA fragments can be varied so as to simulate naturally occurring cell free DNA samples obtained from the blood of the pregnant woman or naturally occurring cell free DNA samples obtained from the blood of cancer patients.
One embodiment is a method of evaluating a fetus for genetic abnormalities (or the risk of genetic abnormalities), comprising, obtaining a blood sample from a pregnant human subject, measuring the fetal fraction of cell free fetal DNA in the blood sample, determining if the fetal fraction of cell free fetal DNA in the blood sample is within the lowest 1 percentile of a reference population, and generating a report indicating that an invasive genetic analysis procedure should be performed. In some embodiments the reference population is matched for gestational age or maternal weight of both gestational age and maternal weight. In some embodiments the test population is matched for gestational age or maternal weight of both gestational age and maternal weight. Genetic abnormalities that can be detected include aneuploidy.
The invention provides methods of increasing the fetal fraction in maternal blood and plasma. This increase in fetal fraction improves the accuracy and decreases the "no call" rate for prenatal testing that measures fetal DNA in maternal blood.
Embodiments include methods, compositions, and kits for creating genetic libraries useful for massively parallel genetic sequencing. Some embodiments are directed to methods of preventing the contamination of genetic libraries with material generated during the formation of other genetic libraries. In some embodiments, the methods employ adapters comprising universal priming sites. The methods can employ non-ligatable primers to generate non-ligatable amplification products so as to prevent unwanted ligation to adapters. In some embodiments, the non-ligatable primers contain uracil. Genetic material can be treated with uracil N glycosylase to prevent the unwanted ligation of uracil containing amplicons to adapters used for creating a second genetic library.
C40B 40/06 - Libraries containing nucleotides or polynucleotides, or derivatives thereof
C40B 40/08 - Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
C40B 30/04 - Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.
The invention provides methods for chromosome copy number calling on genetic samples, such as fetal samples subject to contamination from maternal DNA. The present disclosure provides methods for determining the ploidy status of a chromosome in a fetus (such as a gestating fetus or a POC sample) from genotypic data measured from a mixed sample of DNA comprising DNA from both the mother of the fetus and from the fetus, and optionally from genotypic data from the mother and father. In some embodiments, the ploidy state is determined by using a joint distribution model to create a plurality of expected allele distributions for different possible fetal ploidy states given the parental genotypic data, and comparing the expected allelic distributions to the pattern of measured allelic distributions measured in the mixed sample, and choosing the ploidy state whose expected allelic distribution pattern most closely matches the observed allelic distribution pattern. The mixed sample of DNA may be preferentially enriched at a plurality of polymorphic loci in a way that minimizes the allelic bias, for example using massively multiplexed targeted PCR.
The present disclosure provides methods for determining the ploidy status of an embryo at a chromosome from a sample of DNA from an embryo. The ploidy state is determined by sequencing the DNA from one or more cells biopsied from the embryo, and analyzing the relative amounts of each allele at a plurality of polymorphic loci on the chromosome. In an embodiment, the ploidy state is determined by comparing the observed allele ratios to the expected allele ratios for different ploidy states. In an embodiment, the DNA is selectively amplified at a plurality of polymorphic loci by targeted sequencing. In an embodiment, the mixed sample of DNA may be preferentially enriched at a plurality of polymorphic loci in a way that minimizes the allelic bias.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
G06F 19/20 - for hybridisation or gene expression, e.g. microarrays, sequencing by hybridisation, normalisation, profiling, noise correction models, expression ratio estimation, probe design or probe optimisation
The present disclosure provides methods for determining the ploidy status of a chromosome in a gestating fetus from genotypic data measured from a mixed sample of DNA comprising DNA from both the mother of the fetus and from the fetus, and optionally from genotypic data from the mother and father. The ploidy state is determined by using a joint distribution model to create a plurality of expected allele distributions for different possible fetal ploidy states given the parental genotypic data, and comparing the expected allelic distributions to the pattern of measured allelic distributions measured in the mixed sample, and choosing the ploidy state whose expected allelic distribution pattern most closely matches the observed allelic distribution pattern. The mixed sample of DNA may be preferentially enriched at a plurality of polymorphic loci in a way that minimizes the allelic bias, for example using massively multiplexed targeted PCR.
G06F 19/22 - for sequence comparison involving nucleotides or amino acids, e.g. homology search, motif or Single-Nucleotide Polymorphism [SNP] discovery or sequence alignment
G01N 33/48 - Biological material, e.g. blood, urine; Haemocytometers
40.
METHODS FOR NON-INVASIVE PRENATAL PATERNITY TESTING
Methods for non-invasive prenatal paternity testing are disclosed herein. The method uses genetic measurements made on plasma taken from a pregnant mother, along with genetic measurements of the alleged father, and genetic measurements of the mother, to determine whether or not the alleged father is the biological father of the fetus. This is accomplished by way of an informatics based method that can compare the genetic fingerprint of the fetal DNA found in maternal plasma to the genetic fingerprint of the alleged father.
Methods for non-invasive prenatal ploidy calling are disclosed herein. Methods for determining the ploidy status of a chromosome in a gestating fetus from genotypic data measured from a sample of DNA from the mother of the fetus and from the fetus, and from genotypic data from the mother and optionally also from the father are disclosed herein. The ploidy state is determined by using a joint distribution model to create a set of expected allele distributions for different possible fetal ploidy states given the parental genotypic data, and comparing the expected allelic distributions to the pattern of measured allelic distributions measured in the mixed sample, and choosing the ploidy state whose expected allelic distribution pattern most closely matches the observed allelic distribution pattern. In an embodiment, the mixed sample of DNA may be preferentially enriched at a plurality of polymorphic loci in a way that minimizes the allelic bias.
Disclosed herein are methods for determining the copy number of a chromosome in a fetus in the context of non-invasive prenatal diagnosis. In an embodiment, the measured genetic data from a sample of genetic material that contains both fetal DNA and maternal DNA is analyzed, along with the genetic data from the biological parents of the fetus, and the copy number of the chromosome of interest is determined. In an embodiment, the maternal serum is measured using a single-nucleotide polymorphism (SNP) microarray, along with parental genomic data, and the determination of the chromosome copy number is used to make clinical decisions pertaining to the fetus.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
G01N 33/50 - Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
patents
