The invention relates to methods, compositions, devices, systems and kits are described including, without limitation, reagents and mixtures, for determining the identity of nucleic acids in nucleotide sequences using, for example, data obtained from sequencing by synthesis methods.

Under one aspect, a composition includes a substrate; a first polynucleotide coupled to the substrate; a second polynucleotide hybridized to the first polynucleotide; and a catalyst coupled to a first nucleotide of the second polynucleotide, the catalyst being operable to cause a chemiluminogenic molecule to emit a photon. Under another aspect, a method includes providing a catalyst operable to cause a first chemiluminogenic molecule to emit a photon; providing a substrate; providing a first polynucleotide coupled to the substrate; hybridizing a second polynucleotide to the first polynucleotide; coupling a first quencher to a first nucleotide of the second polynucleotide; and inhibiting, by the first quencher, photon emission by the first chemiluminogenic molecule.

The invention provides a nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker, characterised in that the cleavable linker contains a moiety selected from the group comprising: Formula (I) (wherein X is selected from the group comprising O, S, NH and NQ wherein Q is a C.sub.1-10 substituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl), T is hydrogen or a C.sub.1-10 substituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside).

##STR00001##

The invention provides a nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker, characterised in that the cleavable linker contains a moiety selected from the group comprising: Formula (I) (wherein X is selected from the group comprising O, S, NH and NQ wherein Q is a C.sub.1-10 substituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl), T is hydrogen or a C.sub.1-10 substituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside).

##STR00001##

The present invention provides a method of detecting a heteroresistant population of a pathogen in a sample, the method comprising: a) providing a sample comprising a population of a pathogen; b) extracting nucleic acids from the sample; c) amplifying a target locus of the genome of the pathogen in the extracted nucleic acids, wherein the target locus comprises at least one minor variant associated with drug resistance in the pathogen; d) consecutively sequencing both overlapping nucleic acid strands from a single DNA molecule amplified from the target locus on a Next Generation Sequencing (NGS) platform; e) applying an alignment algorithm to sequencing data from the overlapping nucleic acid strands; and f) performing an analysis of the aligned sequencing data to detect the at least one minor variant and heteroresistant population of the pathogen.

The present invention provides a method of detecting a heteroresistant population of a pathogen in a sample, the method comprising: a) providing a sample comprising a population of a pathogen; b) extracting nucleic acids from the sample; c) amplifying a target locus of the genome of the pathogen in the extracted nucleic acids, wherein the target locus comprises at least one minor variant associated with drug resistance in the pathogen; d) consecutively sequencing both overlapping nucleic acid strands from a single DNA molecule amplified from the target locus on a Next Generation Sequencing (NGS) platform; e) applying an alignment algorithm to sequencing data from the overlapping nucleic acid strands; and f) performing an analysis of the aligned sequencing data to detect the at least one minor variant and heteroresistant population of the pathogen.

In some embodiments, the disclosure relates generally to methods, as well as compositions, systems, kits and apparatuses, for performing nucleotide incorporation, comprising: (a) providing a surface including one or more reaction sites containing a polymerase and a nucleic acid template that has, or is hybridized to, an extendible end; (b) performing a first nucleotide flow by contacting one or more of the reaction sites with a first solution including one or more types of terminator nucleotide; (c) incorporating at least one type of terminator nucleotide at the extendible end of the nucleic acid template contained within at least one of the reaction sites using the polymerase; and (d) detecting a non-optical signal indicating the nucleotide incorporation using a sensor that is attached or operatively linked to the at least one reaction site.

In some embodiments, the disclosure relates generally to methods, as well as compositions, systems, kits and apparatuses, for performing nucleotide incorporation, comprising: (a) providing a surface including one or more reaction sites containing a polymerase and a nucleic acid template that has, or is hybridized to, an extendible end; (b) performing a first nucleotide flow by contacting one or more of the reaction sites with a first solution including one or more types of terminator nucleotide; (c) incorporating at least one type of terminator nucleotide at the extendible end of the nucleic acid template contained within at least one of the reaction sites using the polymerase; and (d) detecting a non-optical signal indicating the nucleotide incorporation using a sensor that is attached or operatively linked to the at least one reaction site.

The invention provides modified nucleotide or nucleoside molecule comprising a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 3-OH blocking group covalently attached thereto, such that the 3 carbon atom has attached a group of the structure OZ wherein Z is any of C(R)2-OR, C(R)2-N(R)2, C(R)2-N(H)R, C(R)2-SR and C(R)2-F, wherein each R is or is part of a removable protecting group; each R is independently a hydrogen atom, an alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy or amido group, or a detectable label attached through a linking group; or (R)2 represents an alkylidene group of formula=C(R)2 wherein each R may be the same or different and is selected from the group comprising hydrogen and halogen atoms and alkyl groups; and wherein said molecule may be reacted to yield an intermediate in which each R is exchanged for H or, where Z is C(R)2-F, the F is exchanged for OH, SH or NH2, preferably OH, which intermediate dissociates under aqueous conditions to afford a molecule with a free 3OH; with the proviso that where Z is C(R)2-SR, both R groups are not H.

The invention provides modified nucleotide or nucleoside molecule comprising a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 3-OH blocking group covalently attached thereto, such that the 3 carbon atom has attached a group of the structure OZ wherein Z is any of C(R)2-OR, C(R)2-N(R)2, C(R)2-N(H)R, C(R)2-SR and C(R)2-F, wherein each R is or is part of a removable protecting group; each R is independently a hydrogen atom, an alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy or amido group, or a detectable label attached through a linking group; or (R)2 represents an alkylidene group of formula=C(R)2 wherein each R may be the same or different and is selected from the group comprising hydrogen and halogen atoms and alkyl groups; and wherein said molecule may be reacted to yield an intermediate in which each R is exchanged for H or, where Z is C(R)2-F, the F is exchanged for OH, SH or NH2, preferably OH, which intermediate dissociates under aqueous conditions to afford a molecule with a free 3OH; with the proviso that where Z is C(R)2-SR, both R groups are not H.