The instant disclosure provides methods of multi-assay processing and multi-assay analysis. Such multi-assay processing and analysis pertain to automated detection of target nucleic acids, e.g., as performed in the clinical setting for diagnostic purposes. Also provided are common assay timing protocols derived from a variety of individual nucleic acid amplification and analysis protocols and modified to prevent resource contention. The instant disclosure also provides systems and devices for practicing the methods as described herein.

Apparatus and method for detecting analyte in a reaction mixture comprising an internal control. The invention is illustrated using amplification and detection of nucleic acids, where the amplification reaction comprises an exogenous internal control. Magnitude of the detection signal serves as the variable for identifying invalid trials, identifying valid trials that are negative for analyte, and identifying trials that are positive for analyte. Detection of a signal indicating probe hybridization can be used for assigning the presence or absence of analyte nucleic acid in validated reactions using only a single detectable label, and without distinguishing the proportion of signal contributed by internal control probe binding, and analyte probe binding.

Apparatus and method for detecting analyte in a reaction mixture comprising an internal control. The invention is illustrated using amplification and detection of nucleic acids, where the amplification reaction comprises an exogenous internal control. Magnitude of the detection signal serves as the variable for identifying invalid trials, identifying valid trials that are negative for analyte, and identifying trials that are positive for analyte. Detection of a signal indicating probe hybridization can be used for assigning the presence or absence of analyte nucleic acid in validated reactions using only a single detectable label, and without distinguishing the proportion of signal contributed by internal control probe binding, and analyte probe binding.

A method for determining the number of nucleic acid molecules (NAMs) in a group of NAMs, comprising i) obtaining an amplified and mutagenized group of NAMs that was produced by a. subjecting the group of NAMs to a chemical mutagenesis which mutates only select nucleic acid bases in the group of NAMs at a rate of 10% to 90% thus forming a group of mutagenized NAMs (mNAMs), and b. amplifying the group of mNAMs; ii) obtaining sequences of the mNAMs in the group of amplified mNAMs; and iii) counting the number of different sequences obtained in step (ii) to determine the number of unique mNAMs in the group of mNAMS,
thereby determining the number of NAMs in the group of NAMs.

A method for determining the number of nucleic acid molecules (NAMs) in a group of NAMs, comprising i) obtaining an amplified and mutagenized group of NAMs that was produced by a. subjecting the group of NAMs to a chemical mutagenesis which mutates only select nucleic acid bases in the group of NAMs at a rate of 10% to 90% thus forming a group of mutagenized NAMs (mNAMs), and b. amplifying the group of mNAMs; ii) obtaining sequences of the mNAMs in the group of amplified mNAMs; and iii) counting the number of different sequences obtained in step (ii) to determine the number of unique mNAMs in the group of mNAMS,
thereby determining the number of NAMs in the group of NAMs.

Particular aspects provide novel, high-throughput methods to quantify DNA methylation (e.g., at a single-base resolution) in an allele-specific manner. The methods comprise use of an allele-specific sequence polymorphism (e.g., allele-specific single nucleotide polymorphism; SNP) in sufficient proximity to a CpG methylation site to provide for distinguishing the methylation levels between two alleles. In particular aspects, after bisulfite modification, the genomic DNA region is PCR-amplified, and the product subjected to allele-specific pyrosequencing, and the percentage of methylation determined based on the percentage of cytosine to thymidine conversion. In further embodiments, MethyLight™ is used after bisulfite treatment. The inventive methodology has, for example, substantial utility for affording quantitative analyses in the regulation of analyses of X-inactivation, the allele-specific expression of genes (e.g., in the immune system) and junk DNA, etc., and in classifying an individual as to whether they have loss of imprinting (LOI).

Particular aspects provide novel, high-throughput methods to quantify DNA methylation (e.g., at a single-base resolution) in an allele-specific manner. The methods comprise use of an allele-specific sequence polymorphism (e.g., allele-specific single nucleotide polymorphism; SNP) in sufficient proximity to a CpG methylation site to provide for distinguishing the methylation levels between two alleles. In particular aspects, after bisulfite modification, the genomic DNA region is PCR-amplified, and the product subjected to allele-specific pyrosequencing, and the percentage of methylation determined based on the percentage of cytosine to thymidine conversion. In further embodiments, MethyLight™ is used after bisulfite treatment. The inventive methodology has, for example, substantial utility for affording quantitative analyses in the regulation of analyses of X-inactivation, the allele-specific expression of genes (e.g., in the immune system) and junk DNA, etc., and in classifying an individual as to whether they have loss of imprinting (LOI).

The method for analyzing biomolecules, includes the steps of: immobilizing biomolecules to be analyzed on surfaces of magnetic microparticles; reacting labeled probe molecules with the biomolecules to be analyzed; collecting and immobilizing the microparticles on a support substrate; and measuring a label on the support substrate. Since single-molecule immobilized magnetic microparticles are used in the present invention, the number of biomolecules can be counted, and since hybridization and an antigen-antibody reaction are performed with the microparticles having biomolecules immobilized thereon dispersed, the reaction can be rapidly performed. Further, the type and the abundance of biomolecules of interest can be determined at a single molecular level, so as to evaluate, in particular, the absolute concentration of biomolecules.

The method for analyzing biomolecules, includes the steps of: immobilizing biomolecules to be analyzed on surfaces of magnetic microparticles; reacting labeled probe molecules with the biomolecules to be analyzed; collecting and immobilizing the microparticles on a support substrate; and measuring a label on the support substrate. Since single-molecule immobilized magnetic microparticles are used in the present invention, the number of biomolecules can be counted, and since hybridization and an antigen-antibody reaction are performed with the microparticles having biomolecules immobilized thereon dispersed, the reaction can be rapidly performed. Further, the type and the abundance of biomolecules of interest can be determined at a single molecular level, so as to evaluate, in particular, the absolute concentration of biomolecules.

The present invention relates to methods of imaging template hybridisation for estimating cluster numbers prior to solid phase amplification and sequencing. More particularly, an initial round of imaging is carried out at the single molecule template hybridisation stage which allows a general estimation of cluster numbers prior to clusters being formed. Amplification of the signal allows single molecule imaging to be carried out using standard sequencing imaging apparatus.