Disclosed are methods for determining copy number variation (CNV) known or suspected to be associated with a variety of medical conditions. In some embodiments, methods are provided for determining copy number variation of fetuses using maternal samples comprising maternal and fetal cell free DNA. In some embodiments, methods are provided for determining CNVs known or suspected to be associated with a variety of medical conditions. Some embodiments disclosed herein provide methods to improve the sensitivity and/or specificity of sequence data analysis by deriving a fragment size parameter. In some implementations, information from fragments of different sizes are used to evaluate copy number variations. In some implementations, one or more t-statistics obtained from coverage information of the sequence of interest is used to evaluate copy number variations. In some implementations, one or more fetal fraction estimates are combined with one or more t-statistics to determine copy number variations.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
G06F 19/24 - for machine learning, data mining or biostatistics, e.g. pattern finding, knowledge discovery, rule extraction, correlation, clustering or classification
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
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
G06F 19/00 - Digital computing or data processing equipment or methods, specially adapted for specific applications (specially adapted for specific functions G06F 17/00;data processing systems or methods specially adapted for administrative, commercial, financial, managerial, supervisory or forecasting purposes G06Q;healthcare informatics G16H)
2.
USING CELL-FREE DNA FRAGMENT SIZE TO DETERMINE COPY NUMBER VARIATIONS
Disclosed are methods for determining copy number variation (CNV) known or suspected to be associated with a variety of medical conditions. In some embodiments, methods are provided for determining copy number variation (CNV) of fetuses using maternal samples comprising maternal and fetal cell free DNA. In some embodiments, methods are provided for determining CNVs known or suspected to be associated with a variety of medical conditions. Some embodiments disclosed herein provide methods to improve the sensitivity and/or specificity of sequence data analysis by deriving a fragment size parameter, such as a size-weighted coverage or a fraction of fragments in a size range. In some embodiments, the fragment size parameter is adjusted to remove within-sample GC-content bias. In some embodiments, removal of within-sample GC-content bias is based on sequence data corrected for systematic variation common across unaffected training samples. Also disclosed are systems and computer program products for evaluation of CNV of sequences of interest.
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
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
3.
DETECTING FETAL SUB-CHROMOSOMAL ANEUPLOIDIES AND COPY NUMBER VARIATIONS
Disclosed are methods for determining copy number variation (CNV) known or suspected to be associated with a variety of medical conditions, including syndromes related to CNV of subchromosomal regions wherein the bins from the unaffected training samples used as controls have a coverage similar to the coverage of the region inspected for CNV. In some embodiments, methods are provided for determining CNV of fetuses using maternal samples comprising maternal and fetal cell free DNA. Some embodiments disclosed herein provide methods to improve the sensitivity and/or specificity of sequence data analysis by removing within-sample GC-content bias. In some embodiments, removal of within-sample GC-content bias is based on sequence data corrected for systematic variation common across unaffected training samples. In some embodiments, syndrome related biases in sample data are also removed to increase signal to noise ratio. Also disclosed are systems for evaluation of CNV of sequences of interest.
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
4.
METHOD FOR IMPROVING THE SENSITIVITY OF DETECTION IN DETERMINING COPY NUMBER VARIATIONS
Disclosed are methods for determining copy number variation (CNV) known or suspected to be associated with a variety of medical conditions. In some embodiments, methods are provided for determining copy number variation (CNV) of fetuses using maternal samples comprising maternal and fetal cell free DNA. In some embodiments, methods are provided for determining CNVs known or suspected to be associated with a variety of medical conditions. Some embodiments disclosed herein provide methods to improve the sensitivity and/or specificity of sequence data analysis by removing within-sample GC-content bias. In some embodiments, removal of within-sample GC-content bias is based on sequence data corrected for systematic variation common across unaffected training samples. Also disclosed are systems and computer program products for evaluation of CNV of sequences of interest.
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
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
5.
METHOD FOR DETERMINING COPY NUMBER VARIATIONS IN SEX CHROMOSOMES
The invention provides methods for determining copy number of the Y chromosome, including, but not limited to, methods for gender determination or Y chromosome aneuploidy of fetus using maternal samples comprising maternal and fetal cell free DNA. Some embodiments disclosed herein describe a strategy for filtering out (or masking) non-discriminant sequence reads on chromosome Y using representative training set of female samples. In some embodiments, this filtering strategy is also applicable to filtering autosomes for evaluation of copy number variation of sequences on the autosomes. In some embodiments, methods are provided for determining copy number variation (CNV) of any fetal aneuploidy, and CNVs known or suspected to be associated with a variety of medical conditions. Also disclosed are systems for evaluation of CNV of sequences of interest on the Y chromosome and other chromosomes.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
G06F 19/24 - for machine learning, data mining or biostatistics, e.g. pattern finding, knowledge discovery, rule extraction, correlation, clustering or classification
6.
GENERATING CELL-FREE DNA LIBRARIES DIRECTLY FROM BLOOD
The disclosure provides methods and kits for preparing sequencing library to detect chromosomal abnormality using cell-free DNA (cfDNA) without the need of first isolating the cfDNA from a liquid fraction of a test sample. In some embodiments, the method involves reducing the binding between the cfDNA and nucleosomal proteins without unwinding the cfDNA from the nucleosomal proteins. In some embodiments, the reduction of binding may be achieved by treating with a detergent or heating. In some embodiments, the method further involves freezing and thawing the test sample before reducing the binding between the cfDNA and the nucleosomal proteins. In some embodiments, the test sample is a peripheral blood sample from a pregnant woman including cfDNA of both a mother and a fetus, wherein the methods may be used to detect fetal chromosomal abnormality such as copy number variation. In other embodiments, the test sample is a peripheral blood sample from a patient known or suspected to have cancer, wherein the methods can be used to detect chromosomal abnormalities in the cfDNA of the patient. Kits for detection of copy number variation of the fetus using the disclosed methods are also provided.
The invention provides a method for determining copy number variations (CNV) of a sequence of interest in a test sample that comprises a mixture of nucleic acids that are known or are suspected to differ in the amount of one or more sequence of interest. The method comprises a statistical approach that accounts for accrued variability stemming from process-related, interchromosomal and inter-sequencing variability. The method is applicable to determining CNV of any fetal aneuploidy, and CNVs known or suspected to be associated with a variety of medical conditions. CNV that can be determined accord ing to the method include trisomies and monosomies of any one or more of chromosomes 1-22, X and Y, other chromosomal polysomies, and deletions and/or duplications of segments of any one or more of the chromosomes, which can be detected by sequencing only once the nucleic acids of a test sample.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
G06F 19/22 - for sequence comparison involving nucleotides or amino acids, e.g. homology search, motif or Single-Nucleotide Polymorphism [SNP] discovery or sequence alignment
The invention provides a method for determining copy number variations (CNV) of a sequence of interest in a test sample that comprises a mixture of nucleic acids that are known or are suspected to differ in the amount of one or more sequence of interest. The method comprises a statistical approach that accounts for accrued variability stemming from process-related, interchromosomal and inter-sequencing variability. The method is applicable to determining CNV of any fetal aneuploidy, and CNVs known or suspected to be associated with a variety of medical conditions. CNV that can be determined accord ing to the method include trisomies and monosomies of any one or more of chromosomes 1 -22, X and Y, other chromosomal polysomies, and deletions and/or duplications of segments of any one or more of the chromosomes, which can be detected by sequencing only once the nucleic acids of a test sample.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
G06F 19/22 - for sequence comparison involving nucleotides or amino acids, e.g. homology search, motif or Single-Nucleotide Polymorphism [SNP] discovery or sequence alignment
9.
DETECTING AND CLASSIFYING COPY NUMBER VARIATION IN A FETAL GENOME
The invention provides a method for determining copy number variations (CNV) of a sequence of interest in a test sample that comprises a mixture of nucleic acids that are known or are suspected to differ in the amount of one or more sequence of interest. The method comprises a statistical approach that accounts for accrued variability stemming from process-related, interchromosomal and inter-sequencing variability. The method is applicable to determining CNV of any fetal aneuploidy, and CNVs known or suspected to be associated with a variety of medical conditions. CNV that can be determined according to the method include trisomies and monosomies of any one or more of chromosomes 1-22, X and Y, other chromosomal polysomies, and deletions and/or duplications of segments of any one or more of the chromosomes, which can be detected by sequencing only once the nucleic acids of a test sample.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
G06F 19/22 - for sequence comparison involving nucleotides or amino acids, e.g. homology search, motif or Single-Nucleotide Polymorphism [SNP] discovery or sequence alignment
10.
SET MEMBERSHIP TESTERS FOR ALIGNING NUCLEIC ACID SAMPLES
Disclosed are methods and tools for rapidly aligning reads to a reference sequence. These methods and tools employ Bloom filters or similar set membership testers to perform the alignment. The reads may be short sequences of nucleic acids or other biological molecules and the reference sequences may be sequences of genomes, chromosomes, etc. The Bloom filters include a collection of hash functions, a bit array, and associated logic for applying reads to the filter. Each filter, and there may be multiple of these used in a particular application, is used to determine whether an applied read is present in a reference sequence. Each Bloom filter is associated with a single reference sequence such as the sequence of a particular chromosome. In one example, chromosomal abundance is determined by aligning reads from a sequencer to multiple chromosomes, each having an associated Bloom filter or other set membership tester.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
G06F 19/22 - for sequence comparison involving nucleotides or amino acids, e.g. homology search, motif or Single-Nucleotide Polymorphism [SNP] discovery or sequence alignment
11.
METHOD FOR DETERMINING THE PRESENCE OR ABSENCE OF DIFFERENT ANEUPLOIDIES IN A SAMPLE
ABSTRACT OF THE DISCLOSURE The invention provides a method for determining copy number variations (CNV) of a sequence of interest in a test sample that comprises a mixture of nucleic acids that are known or are suspected to differ in the amount of one or more sequence of interest. The method comprises a statistical approach that accounts for accrued variability stemming from process-related, interchromosomal and inter-sequencing variability. The method is applicable to determining CNV of any fetal aneuploidy, and CNVs known or suspected to be associated with a variety of medical conditions. CNV that can be determined according to the present method include trisomies and monosomies of any one or more of chromosomes 1-22, X and Y, other chromosomal polysomies, and deletions and/or duplications of segments of any one or more of the chromosomes, which can be detected by sequencing only once the nucleic acids of a test sample. Any aneuploidy can be determined from sequencing information that is obtained by sequencing only once the nucleic acids of a test sample.
The present invention provides a method capable of detecting single or multiple fetal chromosomal aneuploidies in a maternal sample comprising fetal and maternal nucleic acids, and verifying that the correct determination has been made. The method is applicable to determining copy number variations (CNV) of any sequence of interest in samples comprising mixtures of genomic nucleic acids derived from two different genomes, and which are known or are suspected to differ in the amount of one or more sequence of interest. The method is applicable at least to the practice of noninvasive prenatal diagnostics, and to the diagnosis and monitoring of conditions associated with a difference in sequence representation in healthy versus diseased individuals.
Methods of reliably estimating genomic fraction (e.g., fetal fraction) from polymorphisms such as small base variations or insertions-deletions are disclosed. Sequenced data from a multigenomic source is used to determine allele counts for one or more of the polymorphisms. For one or more of the polymorphisms, zygosity is assigned, and genomic fraction is determined from the zygosity and allele counts. Certain embodiments employ SNPs as the relevant polymorphism. The disclosed methods can be applied as part of an intentional, pre-designed re-sequencing study targeted against known polymorphisms or can be used in a retrospective analysis of variations found by coincidence in overlapping sequences generated from maternal plasma (or any other setting where a mixture of DNA from several people are present).
The present invention relates to a method for verifying the integrity of biological source samples subjected to multistep bioassays that comprise massively parallel sequencing of she sample genomic nucleic acids. The integrity of the biological source samples is verified using unique marker nucleic acids that are combined with the biological source sample, and are sequenced concomitantly with the genomic nucleic acids of the biological source sample. The method provides verification of individual samples in single and multiplex massively parallel sequencing assays.
The invention provides a method for determining copy number variations (CNV) of a sequence of interest in a test sample that comprises a mixture of nucleic acids that are known or are suspected to differ in the amount of one or more sequence of interest. The method comprises a statistical approach that accounts for accrued variability stemming from process-related, interchromosomal and inter-sequencing variability. The method is applicable to determining CNV of any fetal aneuploidy, and CNVs known or suspected to be associated with a variety of medical conditions.
The present invention relates to methods comprising whole genome sequencing for identifying polymorphisms in samples comprising mixtures of genomes, and for determining and/or monitoring the presence or absence of disorders associated with the identified polymorphisms.
The invention provides compositions and methods for determining the fraction of fetal nucleic acids in a maternal sample comprising a mixture of fetal and maternal nucleic acids. The fraction of fetal nucleic acids can be used in determining the presence or absence of fetal aneuploidy.
The invention provides compositions and methods for simultaneously determining the presence or absence of fetal aneuploidy and the relative amount of fetal nucleic acids in a sample obtained form a pregnant female. The method encompasses the use of sequencing technologies and exploits the occurrence of polymorphisms to provide a streamlined noninvasive process applicable to the practice of prenatal diagnostics.
The invention provides methods for determining aneuploidy and/or fetal fraction in maternal samples comprising fetal and maternal cfDNA by massively parallel sequencing. The method comprises a novel protocol for preparing sequencing libraries that unexpectedly improves the quality of library DNA while expediting the process of analysis of samples for prenatal diagnoses.
Methods are disclosed for resolving measurement problems such problems in measuring chromosomal copy number. Some disclosed methods involve first selecting a primary assay element characteristic to partition. Such characteristic may be a source of experimental variability such as the GC content of measured DNA sequences. Additionally, the disclosed methods may employ an abundance or copy number function to transform the assay element frequencies into an abundance, dose, copy number score, or the like. In some cases, the disclosed methods estimate an amount of certain fetal DNA in a sample. The methods can further compare the estimated amount to a measured amount of fetal DNA in the sample. The comparison can be used to determine the fetal sex or aneuploidy.
G01N 31/00 - Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroups; Apparatus specially adapted for such methods
Fragile cells have value for use in diagnosing many types of conditions. There is a need for compositions that stabilize fragile cells. The stabilization compositions of the provided invention allow for the stabilization, enrichment, and analysis of fragile cells, including fetal cells, circulating tumor cells, and stem cells.
The present invention provides methods and compositions for specifically identifying a fetal cell. An initial screening of approximately 400 candidate genes by digital PCR in different fetal and adult tissues identified a subset of 24 gene markers specific for fetal nucleated RBC and trophoblasts. The specific expression of those genes was further evaluated and verified in more defined tissues and isolated cells through quantitative RT-PCR using custom Taqman probes specific for each gene. A subset of fetal cell specific markers (FCM) was tested and validated by RNA fluorescent in situ hybridization (FISH) in blood samples from non-pregnant women, and pre-termination and post-termination pregnant women. Applications of these gene markers include, but are not limited to, distinguishing a fetal cell from a maternal cell for fetal cell identification and genetic diagnosis, identifying circulating fetal cell types in maternal blood, purifying or enriching one or more fetal cells, and enumerating one or more fetal cells during fetal cell enrichment.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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
patents
