Provided herein are methods, systems, and compositions for determining a base in a polynucleotide. In various aspects, the methods, systems, and compositions presented herein are useful for performing 4-base, 5-base, or 6-base sequencing of polynucleotide molecules, for example, from liquid biopsy samples or wherein the base is a low frequency mutation.

Provided herein are methods, systems, and compositions for determining a base in a polynucleotide. In various aspects, the methods, systems, and compositions presented herein are useful for performing 4-base, 5-base, or 6-base sequencing of polynucleotide molecules, for example, from liquid biopsy samples or wherein the base is a low frequency mutation.

Methods of characterizing an analyte using a nanopore. One aspect features methods for characterizing a double-stranded polynucleotide using a nanopore, e.g., without using a hairpin connecting a template and a complement of the double-stranded polynucleotide. Another aspect features methods for characterizing an analyte using a tag-modified nanopore with increased sensitivity and/or higher throughput. Compositions and systems including, e.g., adaptors for attachment to double-stranded polynucleotides and tag-modified nanopores, which can be used in the methods are also provided.

Methods of characterizing an analyte using a nanopore. One aspect features methods for characterizing a double-stranded polynucleotide using a nanopore, e.g., without using a hairpin connecting a template and a complement of the double-stranded polynucleotide. Another aspect features methods for characterizing an analyte using a tag-modified nanopore with increased sensitivity and/or higher throughput. Compositions and systems including, e.g., adaptors for attachment to double-stranded polynucleotides and tag-modified nanopores, which can be used in the methods are also provided.

The invention relates to a new method of characterizing a target polynucleotide. The method uses a pore and a Hel308 helicase or amolecular motor which is capable of binding to the target polynucleotide at an internal nucleotide. The helicase or molecular motor controls the movement of the target polynucleotide through the pore.

The invention relates to a new method of characterizing a target polynucleotide. The method uses a pore and a Hel308 helicase or amolecular motor which is capable of binding to the target polynucleotide at an internal nucleotide. The helicase or molecular motor controls the movement of the target polynucleotide through the pore.

Molecular-based diagnostic kits and methods for detecting nucleotide sequences of DNA, DNA copy and RNA of infectious agents, transgenes, alleles or non-encoding sequences, with no initial isolation are provided. The kit comprises at least a first calibrated dropper bottle with an anti-contamination system containing a solution of polymerase enzyme, chaotropic agents, salts and deoxyribonucleotides; a second calibrated dropper bottle with an anti-contamination system containing a set of at least 4 primers, part of the sequences being preserved in the nucleic acid fragment to be detected; a dropper bottle containing a mixture of developing reagents; a carrier to hold the sample; at least one LFD developing system, colorimetric or fluorogenic reaction; positive and negative controls. The polymerase enzyme is selected from Bst enzymes formed by DNA polymerase/helicase, with no exonuclease activity. The set of primers is designed from gene sequences to be detected, whether of deoxyribonucleic or ribonucleic nature.

Molecular-based diagnostic kits and methods for detecting nucleotide sequences of DNA, DNA copy and RNA of infectious agents, transgenes, alleles or non-encoding sequences, with no initial isolation are provided. The kit comprises at least a first calibrated dropper bottle with an anti-contamination system containing a solution of polymerase enzyme, chaotropic agents, salts and deoxyribonucleotides; a second calibrated dropper bottle with an anti-contamination system containing a set of at least 4 primers, part of the sequences being preserved in the nucleic acid fragment to be detected; a dropper bottle containing a mixture of developing reagents; a carrier to hold the sample; at least one LFD developing system, colorimetric or fluorogenic reaction; positive and negative controls. The polymerase enzyme is selected from Bst enzymes formed by DNA polymerase/helicase, with no exonuclease activity. The set of primers is designed from gene sequences to be detected, whether of deoxyribonucleic or ribonucleic nature.

An example method includes reacting a first solution and a different, second solution on a flow cell by flowing the first solution over amplification sites on the flow cell and subsequently flowing the second solution over the amplification sites. The first solution includes target nucleic acids and a first reagent mixture that comprises nucleoside triphosphates and replication enzymes. The target nucleic acids in the first solution transport to and bind to the amplification sites at a transport rate. The first reagent mixture amplifies the target nucleic acids that are bound to the amplification sites to produce clonal populations of amplicons originating from corresponding target nucleic acids. The amplicons are produced at an amplification rate that exceeds the transport rate. The second solution includes a second reagent mixture and lacks the target nucleic acids. The second solution is to increase a number of the amplicons at the amplification sites.

An example method includes reacting a first solution and a different, second solution on a flow cell by flowing the first solution over amplification sites on the flow cell and subsequently flowing the second solution over the amplification sites. The first solution includes target nucleic acids and a first reagent mixture that comprises nucleoside triphosphates and replication enzymes. The target nucleic acids in the first solution transport to and bind to the amplification sites at a transport rate. The first reagent mixture amplifies the target nucleic acids that are bound to the amplification sites to produce clonal populations of amplicons originating from corresponding target nucleic acids. The amplicons are produced at an amplification rate that exceeds the transport rate. The second solution includes a second reagent mixture and lacks the target nucleic acids. The second solution is to increase a number of the amplicons at the amplification sites.