Real time electronic sequencing devices, chips, and systems are described. Arrays of nanoFET devices are used to provide sequence information about a template nucleic acid in a polymerase-template complex bound to the nanoFET. The nanoFET devices typically have a source, a drain and a gate comprising a nanowire. A single polymerase enzyme complex comprising a polymerase enzyme complexed with the template nucleic acid is bound to the gate. The polymerase is bound to the gate non-covalently through a polymeric binding agent that has two strands, each strand interacting with the nanowire such that the polymerase is in a central location between the strands with the polymeric binding agent extending away from the polymerase complex along the nanowire in both directions.

In some aspects, the present disclosure provides methods for forming ligation products comprising single-stranded polynucleotides without the need for enriching the single-stranded polynucleotides. Ligation products formed by various aspects of the present disclosure can be useful for various applications, including but not limited to sequence analysis. In some embodiments, the ligation products comprise cell-free polynucleotides. In some aspects, the present disclosure provides reaction mixtures, kits and complexes consistent with the methods herein.

In some aspects, the present disclosure provides methods for forming ligation products comprising single-stranded polynucleotides without the need for enriching the single-stranded polynucleotides. Ligation products formed by various aspects of the present disclosure can be useful for various applications, including but not limited to sequence analysis. In some embodiments, the ligation products comprise cell-free polynucleotides. In some aspects, the present disclosure provides reaction mixtures, kits and complexes consistent with the methods herein.

The present disclosure provide systems, compositions, methods, reagents, kits and products for extending a nucleic acid that includes incorporating a nucleotide residue at a terminus of a nucleic acid using a polymerase enzyme and at least one nucleotide, wherein the at least one nucleotide includes a thiophosphate moiety, and wherein the at least one nucleotide is resistant to hydrolysis by phosphatase. In some embodiments, the nucleotide incorporation can be conducted in the presence of a phosphatase. In some embodiments, the nucleotide incorporation can be conducted in the presence of at least on chelation moiety that is configured to bind an orthophosphate moiety.

The present disclosure provide systems, compositions, methods, reagents, kits and products for extending a nucleic acid that includes incorporating a nucleotide residue at a terminus of a nucleic acid using a polymerase enzyme and at least one nucleotide, wherein the at least one nucleotide includes a thiophosphate moiety, and wherein the at least one nucleotide is resistant to hydrolysis by phosphatase. In some embodiments, the nucleotide incorporation can be conducted in the presence of a phosphatase. In some embodiments, the nucleotide incorporation can be conducted in the presence of at least on chelation moiety that is configured to bind an orthophosphate moiety.

A method of detecting a target polynucleotide sequence in a given nucleic acid analyte characterised by the steps of: a. annealing the analyte to a single-stranded probe oligonucleotide A.sub.0 to create a first intermediate product which is at least partially double-stranded and in which the 3′ end of A.sub.0 forms a double-stranded complex with the analyte target sequence; b. pyrophosphorolysing the first intermediate product with a pyrophosphorolysing enzyme in the 3′-5′ direction from the 3′ end of A.sub.0 to create partially digested strand A.sub.1 and the analyte; c. (i) annealing A.sub.1 to a single-stranded trigger oligonucleotide B and extending the A.sub.1 strand in the 5′-3′ direction against B; or (ii) circularising A.sub.1 through ligation of its 3′ and 5′ ends; or (iii) ligating the 3′ end of A.sub.1 to the 5′ end of a ligation probe oligonucleotide C; in each case to create an oligonucleotide A.sub.2; d. priming A.sub.2 with at least one single-stranded primer oligonucleotide and creating multiple copies of A.sub.2, or a region of A.sub.2; and e. detecting a signal derived from the multiple copies and inferring therefrom the presence or absence of the polynucleotide target sequence in the analyte.

A method of detecting a target polynucleotide sequence in a given nucleic acid analyte characterised by the steps of: a. annealing the analyte to a single-stranded probe oligonucleotide A.sub.0 to create a first intermediate product which is at least partially double-stranded and in which the 3′ end of A.sub.0 forms a double-stranded complex with the analyte target sequence; b. pyrophosphorolysing the first intermediate product with a pyrophosphorolysing enzyme in the 3′-5′ direction from the 3′ end of A.sub.0 to create partially digested strand A.sub.1 and the analyte; c. (i) annealing A.sub.1 to a single-stranded trigger oligonucleotide B and extending the A.sub.1 strand in the 5′-3′ direction against B; or (ii) circularising A.sub.1 through ligation of its 3′ and 5′ ends; or (iii) ligating the 3′ end of A.sub.1 to the 5′ end of a ligation probe oligonucleotide C; in each case to create an oligonucleotide A.sub.2; d. priming A.sub.2 with at least one single-stranded primer oligonucleotide and creating multiple copies of A.sub.2, or a region of A.sub.2; and e. detecting a signal derived from the multiple copies and inferring therefrom the presence or absence of the polynucleotide target sequence in the analyte.

Disclosed are methods for nucleic acid amplification wherein nucleic acid templates, beads, and amplification reaction solution are emulsified and the nucleic acid templates are amplified to provide clonal copies of the nucleic acid templates attached to the beads. Also disclosed are kits and apparatuses for performing the methods of the invention.

Disclosed are methods for nucleic acid amplification wherein nucleic acid templates, beads, and amplification reaction solution are emulsified and the nucleic acid templates are amplified to provide clonal copies of the nucleic acid templates attached to the beads. Also disclosed are kits and apparatuses for performing the methods of the invention.

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