Provided herein are methods and systems for detecting the presence of absence of a target-nucleic acid sequence, including SARS-COV2.

Methods and apparatus relating to very large scale FET arrays for analyte measurements. ChemFET (e.g., ISFET) arrays may be fabricated using conventional CMOS processing techniques based on improved FET pixel and array designs that increase measurement sensitivity and accuracy, and at the same time facilitate significantly small pixel sizes and dense arrays. Improved array control techniques provide for rapid data acquisition from large and dense arrays. Such arrays may be employed to detect a presence and/or concentration changes of various analyte types in a wide variety of chemical and/or biological processes. In one example, chemFET arrays facilitate DNA sequencing techniques based on monitoring changes in hydrogen ion concentration (pH), changes in other analyte concentration, and/or binding events associated with chemical processes relating to DNA synthesis.

Methods and apparatus relating to very large scale FET arrays for analyte measurements. ChemFET (e.g., ISFET) arrays may be fabricated using conventional CMOS processing techniques based on improved FET pixel and array designs that increase measurement sensitivity and accuracy, and at the same time facilitate significantly small pixel sizes and dense arrays. Improved array control techniques provide for rapid data acquisition from large and dense arrays. Such arrays may be employed to detect a presence and/or concentration changes of various analyte types in a wide variety of chemical and/or biological processes. In one example, chemFET arrays facilitate DNA sequencing techniques based on monitoring changes in hydrogen ion concentration (pH), changes in other analyte concentration, and/or binding events associated with chemical processes relating to DNA synthesis.

A method for nucleic acid sequencing may include disposing a plurality of template nucleic acid molecules in a plurality of defined spaces disposed on a sensor array, at least some of the plurality of template nucleic acid molecules having a sequencing primer and a polymerase operably bound therewith; advancing one or more nucleotide species over the plurality of template nucleic acid molecules with the sequencing primer and the polymerase operably bound therewith; measuring a signal generated by nucleotide incorporations resulting from advancing the one or more nucleotide species; and exposing the plurality of template nucleic acid molecules to a cleaving reagent subsequent to the advancing and measuring. The cleaving reagent can remove labeling reagents attached to the one or more nucleotide species. The advancing and measuring steps can be performed for different orders of the one or more nucleotide species prior to a subsequent exposing of the plurality of template nucleic acid molecules to the cleaving reagent.

A method for nucleic acid sequencing may include disposing a plurality of template nucleic acid molecules in a plurality of defined spaces disposed on a sensor array, at least some of the plurality of template nucleic acid molecules having a sequencing primer and a polymerase operably bound therewith; advancing one or more nucleotide species over the plurality of template nucleic acid molecules with the sequencing primer and the polymerase operably bound therewith; measuring a signal generated by nucleotide incorporations resulting from advancing the one or more nucleotide species; and exposing the plurality of template nucleic acid molecules to a cleaving reagent subsequent to the advancing and measuring. The cleaving reagent can remove labeling reagents attached to the one or more nucleotide species. The advancing and measuring steps can be performed for different orders of the one or more nucleotide species prior to a subsequent exposing of the plurality of template nucleic acid molecules to the cleaving reagent.

A minimal-copy-ratio of templates is a problem in detecting early stage cancer where minimal copies of somatic cancer-specific mutations are targeted in the presence of large copies of wildtype genome DNA, commonly a 1/10,000 or even less minimal-copy-ratios between the mutant target and wildtype control templates. To overcome this problem, delayed pyrophosphorolysis activated polymerization (delayed-PAP) was developed which can delay product accumulation of the wildtype control to a much later time or cycle, such as by 15 cycles or by 30,000 folds. In the multiplex format, delayed-PAP is particularly useful to amplify not only the wildtype control but also mutant target templates accurately and consistently in the minimal-copy-ratio situation.

Methods and apparatus relating to very large scale FET arrays for analyte measurements. ChemFET (e.g., ISFET) arrays may be fabricated using conventional CMOS processing techniques based on improved FET pixel and array designs that increase measurement sensitivity and accuracy, and at the same time facilitate significantly small pixel sizes and dense arrays. Improved array control techniques provide for rapid data acquisition from large and dense arrays. Such arrays may be employed to detect a presence and/or concentration changes of various analyte types in a wide variety of chemical and/or biological processes. In one example, chemFET arrays facilitate DNA sequencing techniques based on monitoring changes in the concentration of inorganic pyrophosphate (PPi), hydrogen ions, and nucleotide triphosphates.

Methods and apparatus relating to very large scale FET arrays for analyte measurements. ChemFET (e.g., ISFET) arrays may be fabricated using conventional CMOS processing techniques based on improved FET pixel and array designs that increase measurement sensitivity and accuracy, and at the same time facilitate significantly small pixel sizes and dense arrays. Improved array control techniques provide for rapid data acquisition from large and dense arrays. Such arrays may be employed to detect a presence and/or concentration changes of various analyte types in a wide variety of chemical and/or biological processes. In one example, chemFET arrays facilitate DNA sequencing techniques based on monitoring changes in the concentration of inorganic pyrophosphate (PPi), hydrogen ions, and nucleotide triphosphates.

Provided is a method of gene sequencing based on a single molecule library preparation on a microwell array chip, the single molecule library being further amplified by PCR on the same microwell array chip, the method including: Step 1: adding a PCR amplification system containing DNA fragments to be tested into the microwell array chip, allowing microwells on the microwell array chip to each individually form reaction spaces and allowing one DNA fragment to be contained in one microwell, Step 1: subjecting the sealed microwell array chip after step 1 to PCR amplification reaction on a PCR machine, Step 3: denaturing amplified double-stranded DNA molecules of the DNA fragment in individual microwell to single-stranded DNA molecules, Step 4: allowing sequencing primer S2 molecules to be paired with the single-stranded DNA molecules of the DNA fragment in individual microwell via annealing, Step 5: the dNTP added into microwells in sequence of dGTP, dCTP, dATP and dTTP is paired with a base under sequencing, with hydrogen ions or pyrophosphate PPi ions released and thus charges of DNA backbones increased, resulting in signal response of a sensor at bottom of the microwell, the signal is recorded and converted into gene sequence information, and Step 6: repeating step 5.

Provided is a method of gene sequencing based on a single molecule library preparation on a microwell array chip, the single molecule library being further amplified by PCR on the same microwell array chip, the method including: Step 1: adding a PCR amplification system containing DNA fragments to be tested into the microwell array chip, allowing microwells on the microwell array chip to each individually form reaction spaces and allowing one DNA fragment to be contained in one microwell, Step 1: subjecting the sealed microwell array chip after step 1 to PCR amplification reaction on a PCR machine, Step 3: denaturing amplified double-stranded DNA molecules of the DNA fragment in individual microwell to single-stranded DNA molecules, Step 4: allowing sequencing primer S2 molecules to be paired with the single-stranded DNA molecules of the DNA fragment in individual microwell via annealing, Step 5: the dNTP added into microwells in sequence of dGTP, dCTP, dATP and dTTP is paired with a base under sequencing, with hydrogen ions or pyrophosphate PPi ions released and thus charges of DNA backbones increased, resulting in signal response of a sensor at bottom of the microwell, the signal is recorded and converted into gene sequence information, and Step 6: repeating step 5.