Methods for preparing sequencing libraries from a DNA-containing test sample, as well as methods for correcting sequencing-derived errors, are provided.

Methods for preparing sequencing libraries from a DNA-containing test sample, as well as methods for correcting sequencing-derived errors, are provided.

Methods, polynucleotides, kits, and reaction mixtures are disclosed for the enriching of short polynucleotide molecules that have a length within a desired target length range. A Type IIS or Type III restriction enzyme is used to cleave polynucleotides at cleavage sites located at a distance from the restriction enzyme recognition sites. For example, a mixture of polynucleotides can be formed by inserting DNA molecules between a recognition site for the restriction enzyme and a region of non-naturally-occurring nucleotides that block cleavage by the restriction enzymes. If a polynucleotide contains a DNA molecule with a length within a target range, then the cleavage site will be within the blocking region, and cleavage will not occur. Polynucleotides containing DNA molecules with lengths outside the target range can be cleaved. By selectively enriching, through PCR or other means, polynucleotides that are intact, a concentrated population of polynucleotides of a target length can be formed.

Methods, polynucleotides, kits, and reaction mixtures are disclosed for the enriching of short polynucleotide molecules that have a length within a desired target length range. A Type IIS or Type III restriction enzyme is used to cleave polynucleotides at cleavage sites located at a distance from the restriction enzyme recognition sites. For example, a mixture of polynucleotides can be formed by inserting DNA molecules between a recognition site for the restriction enzyme and a region of non-naturally-occurring nucleotides that block cleavage by the restriction enzymes. If a polynucleotide contains a DNA molecule with a length within a target range, then the cleavage site will be within the blocking region, and cleavage will not occur. Polynucleotides containing DNA molecules with lengths outside the target range can be cleaved. By selectively enriching, through PCR or other means, polynucleotides that are intact, a concentrated population of polynucleotides of a target length can be formed.

Some aspects of this disclosure relate to the identification of reactive nucleic acids, such as, for example, reactive cellular RNAs. Minimal reactive motif consensus sequences and isolated reactive nucleic acids conforming to such consensus sequences are provided herein. Reactive probes that selectively form covalent bonds with reactive nucleic acids are also provided herein, as are methods of using such probes, for example, for labeling, identification, characterization, and/or quantification of reactive nucleic acids. This disclosure further provides fusion molecules comprising a reactive nucleic acid conjugated to a heterologous molecule of interest, such as a heterologous nucleic acid or protein. Methods and kits for generating and using such fusion proteins to track, detect, and/or quantify molecules of interest in a biological sample are also provided as are expression constructs encoding fusion molecules of reactive nucleic acids and heterologous nucleic acids of interest.

Some aspects of this disclosure relate to the identification of reactive nucleic acids, such as, for example, reactive cellular RNAs. Minimal reactive motif consensus sequences and isolated reactive nucleic acids conforming to such consensus sequences are provided herein. Reactive probes that selectively form covalent bonds with reactive nucleic acids are also provided herein, as are methods of using such probes, for example, for labeling, identification, characterization, and/or quantification of reactive nucleic acids. This disclosure further provides fusion molecules comprising a reactive nucleic acid conjugated to a heterologous molecule of interest, such as a heterologous nucleic acid or protein. Methods and kits for generating and using such fusion proteins to track, detect, and/or quantify molecules of interest in a biological sample are also provided as are expression constructs encoding fusion molecules of reactive nucleic acids and heterologous nucleic acids of interest.

A method of tagged-base DNA sequencing readout on waveguide surfaces includes immobilizing, a surface of a waveguide, a nucleotide fragment, exposing the nucleotide fragment to a first plurality of capped nucleotides, wherein the first plurality of capped nucleotides include a first plurality of nucleotide types, each distinct nucleotide type has a distinct capping agent, and each distinct capping agent has a distinct optical signature, severing base pair connections between the at least a nucleotide fragment and the first plurality of capped nucleotides, wherein the nucleotide fragment remains attached and a first single nucleotide, of the first plurality of capped nucleotides, remains immobilized on a nucleotide binding locus adjacent to the first nucleotide sequence, and detecting a first distinct optical signature of a first distinct capping agent of the first single nucleotide using the waveguide.

A method of tagged-base DNA sequencing readout on waveguide surfaces includes immobilizing, a surface of a waveguide, a nucleotide fragment, exposing the nucleotide fragment to a first plurality of capped nucleotides, wherein the first plurality of capped nucleotides include a first plurality of nucleotide types, each distinct nucleotide type has a distinct capping agent, and each distinct capping agent has a distinct optical signature, severing base pair connections between the at least a nucleotide fragment and the first plurality of capped nucleotides, wherein the nucleotide fragment remains attached and a first single nucleotide, of the first plurality of capped nucleotides, remains immobilized on a nucleotide binding locus adjacent to the first nucleotide sequence, and detecting a first distinct optical signature of a first distinct capping agent of the first single nucleotide using the waveguide.

Methods of detecting the absence or presence of a micro-organism in a sample comprising: contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample, incubating the thus contacted sample under conditions suitable for nucleic acid modifying activity; and specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism. Corresponding kits are also provided.

Apparatus and methods for analyzing single molecule and performing nucleic acid sequencing. An integrated device includes multiple pixels with sample wells configured to receive a sample, which, when excited, emits radiation; at least one element for directing the emission radiation in a particular direction; and a light path along which the emission radiation travels from the sample well toward a sensor. The apparatus also includes an instrument that interfaces with the integrated device. Each sensor may detect emission radiation from a sample in a respective sample well. The instrument includes an excitation light source for exciting the sample in each sample well.