This disclosure provides systems and methods for sequencing nucleic acids using nucleotide analogues and translocation of tags from incorporated nucleotide analogues through a nanopore. In aspects, this disclosure is related to composition, method, and system for sequencing a nucleic acid using tag molecules and detection of translocation through a nanopore of tags released from incorporation of the molecule.

A kit for preparing a nucleic acid sample for analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) is provided, the kit comprising: a lyophilized enzyme composition comprising: micrococcal nuclease; nuclease P1; and bacterial alkaline phosphatase (BAP); and a digestion buffer. Also provided are enzyme compositions and methods of use for rapid, efficient preparation of a nucleic acid sample for analysis by LC-MS/MS, without the need for denaturation of the sample.

A kit for preparing a nucleic acid sample for analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) is provided, the kit comprising: a lyophilized enzyme composition comprising: micrococcal nuclease; nuclease P1; and bacterial alkaline phosphatase (BAP); and a digestion buffer. Also provided are enzyme compositions and methods of use for rapid, efficient preparation of a nucleic acid sample for analysis by LC-MS/MS, without the need for denaturation of the sample.

A method for determining the sequence of a target nucleic acid, including steps of contacting a target nucleic acid with a polymerase to sequentially remove nucleotide triphosphates from the target nucleic acid, wherein the nucleotide triphosphates that are removed have a variety of different base moieties; and distinguishing the different base moieties for the nucleotide triphosphates that are removed. Also provided is a apparatus including a nanopore positioned in a fluid impermeable barrier to form a passage through which a nucleotide triphosphate can pass from a first fluid reservoir to a second fluid reservoir, and a reaction mix in the first fluid reservoir that includes a polymerase, target nucleic acid having two strands, and pyrophosphorolytic concentration of pyrophosphate.

A method for determining the sequence of a target nucleic acid, including steps of contacting a target nucleic acid with a polymerase to sequentially remove nucleotide triphosphates from the target nucleic acid, wherein the nucleotide triphosphates that are removed have a variety of different base moieties; and distinguishing the different base moieties for the nucleotide triphosphates that are removed. Also provided is a apparatus including a nanopore positioned in a fluid impermeable barrier to form a passage through which a nucleotide triphosphate can pass from a first fluid reservoir to a second fluid reservoir, and a reaction mix in the first fluid reservoir that includes a polymerase, target nucleic acid having two strands, and pyrophosphorolytic concentration of pyrophosphate.

Compositions comprising activated topoisomerase adaptors (TOPO-adaptors) and methods of using the activated TOPO-adaptors are provided for preparing a library of target DNA duplexes derived from sample polynucleotides (e.g., DNA, RNA) for the streamlined preparation of a large number of samples. Such libraries may be used for Next Generation Sequencing (NGS).

Compositions comprising activated topoisomerase adaptors (TOPO-adaptors) and methods of using the activated TOPO-adaptors are provided for preparing a library of target DNA duplexes derived from sample polynucleotides (e.g., DNA, RNA) for the streamlined preparation of a large number of samples. Such libraries may be used for Next Generation Sequencing (NGS).

The present disclosure describes a method of adapter ligation to the ends of fragmented double-stranded DNA molecules.

The present disclosure describes a method of adapter ligation to the ends of fragmented double-stranded DNA molecules.

The invention provides methods of isolating a target nucleic acid in a sample. A primer is hybridized to the target. A polymerase and modified nucleotide resistant to nuclease degradation are used to extend the primer to create a modified polynucleotide. The sample is exposed to a nuclease, thereby isolating the modified polynucleotide. Optionally, the target nucleic acid may be further protected by binding a protein in a sequence specific manner to one end of the target nucleic acid to create a protected target nucleic acid resistant to nuclease degradation. Thus, after exposing the sample to a nuclease, the modified polynucleotide and protected target nucleic acid are isolated.