Methods and compositions for detecting genetic instability using digital amplification assays. The methods may be performed in a set of isolated volumes and generally may involve competitive hybridization of a competitor and a probe/primer with a normal allele and one or more mutant alleles of a microsatellite locus. The competitor may be configured to compete similarly with, or to outcompete, the primer/probe for hybridization with the normal allele. The primer/probe may be configured to outcompete the competitor for hybridization with various mutant alleles of the locus that alter the length of the repetitive sequence by different amounts. Isolated volumes in which the primer/probe outcompetes the competitor may be enumerated, and represent one or more of the mutant alleles. The methods may enable diagnosing microsatellite instability and treating a subject based on the diagnosis.

The present disclosure generally relates to artificial controls for genetic sequencing and quantitation assays, which can be used to calibrate a wide variety of genetic sequencing and quantitation methods. For example, the controls disclosed herein can be used to calibrate a wide variety of high throughput sequencing methods (for example, those referred to as next generation sequencing methods). The present disclosure also generally relates to the use of the sequencing controls in a wide variety of applications including, for example, in the calibration of a wide variety of sequencing methods.

The present invention provides an epigenetic haemogram, also referred to as an epigenetic blood cell count that identifies the quantitative, comprehensive picture of cellular composition in a biological sample, wherein advantageously a normalization standard is used. The normalization standard is a nucleic acid molecule comprising at least one marker-region being specific for each of the blood cells to be detected, and at least one control-region being cell-unspecific, wherein said regions are present in the same number of copies on said molecule and/or a natural blood cell sample of known composition. Furthermore, the present invention relates to a kit and the use of a kit for performing the epigenetic assessment of comprehensive, quantitative cellular composition of a biological sample. The biological sample is derived from e.g. a mammalian body fluid, including peripheral, capillary or venous blood samples or subfractions thereof, such as peripheral blood mononuclear cells or peripheral blood monocytes, or a tissue sample, organ sample, or from frozen, dried, embedded, stored or fresh body fluids or tissue samples.

The present invention provides an epigenetic haemogram, also referred to as an epigenetic blood cell count that identifies the quantitative, comprehensive picture of cellular composition in a biological sample, wherein advantageously a normalization standard is used. The normalization standard is a nucleic acid molecule comprising at least one marker-region being specific for each of the blood cells to be detected, and at least one control-region being cell-unspecific, wherein said regions are present in the same number of copies on said molecule and/or a natural blood cell sample of known composition. Furthermore, the present invention relates to a kit and the use of a kit for performing the epigenetic assessment of comprehensive, quantitative cellular composition of a biological sample. The biological sample is derived from e.g. a mammalian body fluid, including peripheral, capillary or venous blood samples or subfractions thereof, such as peripheral blood mononuclear cells or peripheral blood monocytes, or a tissue sample, organ sample, or from frozen, dried, embedded, stored or fresh body fluids or tissue samples.

Methods for controlling for non-systematic error in an amplification-based next generation sequencing (NGS) library preparation are described, which method includes using an internal amplification control (IAC) sharing identical priming sites to a native nucleic acid target template of interest in a NGS library preparation.

Methods for controlling for non-systematic error in an amplification-based next generation sequencing (NGS) library preparation are described, which method includes using an internal amplification control (IAC) sharing identical priming sites to a native nucleic acid target template of interest in a NGS library preparation.

In the present invention, a method for the qualitative genetic characterization and/or gene expression characterization of predetermined cells in a fluid sample containing such cells is provided. The inventive method for the qualitative genetic and/or gene expression characterization of predetermined cells in a fluid sample containing such cells, comprises: a) selectively extracting at least a part of the predetermined cells from the sample forming a cell suspension cs.sub.0; and b) repeating the extraction step a) n times with the same sample of step a), with n1, forming at least one cell suspension cs.sub.n; c) determining a gene expression profile gep.sub.n and/or a first copy number count cnc.sub.0 of at least one DNA and/or RNA with at least a part of the cell suspension cs.sub.0; d) determining at least one further gene expression profile gepn and/or a further copy number count cnc.sub.n of at least one DNA and/or RNA with at least a part of at least one further cell suspension csn; e) calculating the predetermined cells' gene expression profile gep(P) of at least one predetermined DNA and/or RNA by subtracting gepn from gep, and/or the predetermined cells' copy number count cnc(P) of at least one predetermined DNA and/or RNA by subtracting cncn from cnc.sub.0; and f) evaluating the qualitative genetic and/or gene expression characteristics of the predetermined cells from gep(P) and/or cnc(P).

In the present invention, a method for the qualitative genetic characterization and/or gene expression characterization of predetermined cells in a fluid sample containing such cells is provided. The inventive method for the qualitative genetic and/or gene expression characterization of predetermined cells in a fluid sample containing such cells, comprises: a) selectively extracting at least a part of the predetermined cells from the sample forming a cell suspension cs.sub.0; and b) repeating the extraction step a) n times with the same sample of step a), with n1, forming at least one cell suspension cs.sub.n; c) determining a gene expression profile gep.sub.n and/or a first copy number count cnc.sub.0 of at least one DNA and/or RNA with at least a part of the cell suspension cs.sub.0; d) determining at least one further gene expression profile gepn and/or a further copy number count cnc.sub.n of at least one DNA and/or RNA with at least a part of at least one further cell suspension csn; e) calculating the predetermined cells' gene expression profile gep(P) of at least one predetermined DNA and/or RNA by subtracting gepn from gep, and/or the predetermined cells' copy number count cnc(P) of at least one predetermined DNA and/or RNA by subtracting cncn from cnc.sub.0; and f) evaluating the qualitative genetic and/or gene expression characteristics of the predetermined cells from gep(P) and/or cnc(P).

The present disclosure generally relates to artificial controls for genetic sequencing and quantitation assays, which can be used to calibrate a wide variety of genetic sequencing and quantitation methods. For example, the controls disclosed herein can be used to calibrate a wide variety of high throughput sequencing methods (for example, those referred to as next generation sequencing methods). The present disclosure also generally relates to the use of the sequencing controls in a wide variety of applications including, for example, in the calibration of a wide variety of sequencing methods.

A method of identifying a first target nucleic acid comprising, providing a sample comprising the first target nucleic acid, providing a first set of paired oligonucleotides with complementarity to the first target nucleic acid, the first set of paired oligonucleotides comprising a first ratio of (a) first competitive oligonucleotides to (b) first signal oligonucleotides comprising a signal tag, wherein the competitive oligonucleotides compete with the signal oligonucleotides for binding to the first target nucleic acid, amplifying the first target nucleic acid with the polymerase chain reaction, thereby degrading the first signal oligonucleotide and permitting generation of a first signal, generating the first signal, measuring intensity of the first signal, and correlating the intensity of the first signal to the first ratio, thereby identifying the first target nucleic acid.