Methods are provided that, for example, include (a) combining ssDNA containing a modified nucleotide (e.g., a ssDNA with a modified nucleotide proximate to its 5′ end) with a DNA cleavage enzyme capable of cleaving the ssDNA at the modified nucleotide (e.g., to generate a first ssDNA fragment having a 3′OH and a second ssDNA fragment having the modified nucleotide); wherein the ratio of enzyme to DNA substrate is less than 1:1 molar ratio (m/m); and (b) cleaving at least 95% of the ssDNA at the modified nucleotide. In some embodiments, a method may comprise (a) combining (i) a ssDNA comprising a modified nucleotide (e.g., proximate to its 5′ end) with (ii) a DNA cleavage enzyme capable of cleaving the ssDNA at the modified nucleotide (e.g., to generate (after cleavage) a first ssDNA fragment having a 3′OH and a second ssDNA fragment comprising the modified nucleotide) wherein the ratio of enzyme to DNA substrate is less than 1:1 molar ratio and cleaving at least 95% of the ssDNA at the modified nucleotide. In some embodiments, methods provided herein may include (a) combining (i) a ssDNA (1) immobilized on a substrate and (2) comprising a modified nucleotide with (ii) a ssDNA cleaving enzyme capable of cleaving the ssDNA at the modified nucleotide (e.g., to generate (after cleavage) a first ssDNA fragment having a 3′OH and a second ssDNA fragment comprising the modified nucleotide) ; and (b) cleaving the immobilized ssDNA to release the second single stranded DNA fragment from the substrate. At least 95% (m/m) of an ssDNA comprising a modified nucleotide may be cleaved in less than 60 minutes.

Methods and systems for digital measurements are provided. In an embodiment, the method includes producing a plurality of droplets, wherein at least one of the droplets of the plurality of droplets contains an analyte molecule from a sample; measuring at least a first portion of the plurality of droplets to determine individual volumes of droplets in the first portion of the plurality of droplets; analyzing at least a second portion of the plurality of droplets to determine a number of droplets in the second portion of the plurality of droplets that contain the analyte molecule; and using individual volumes of the droplets in the first portion of the plurality of droplets and the number of droplets in the second portion of the plurality of droplets that contain the analyte molecule to determine the concentration of the analyte molecule in the sample.

Methods and systems for digital measurements are provided. In an embodiment, the method includes producing a plurality of droplets, wherein at least one of the droplets of the plurality of droplets contains an analyte molecule from a sample; measuring at least a first portion of the plurality of droplets to determine individual volumes of droplets in the first portion of the plurality of droplets; analyzing at least a second portion of the plurality of droplets to determine a number of droplets in the second portion of the plurality of droplets that contain the analyte molecule; and using individual volumes of the droplets in the first portion of the plurality of droplets and the number of droplets in the second portion of the plurality of droplets that contain the analyte molecule to determine the concentration of the analyte molecule in the sample.

The present invention features compositions and methods for quantifying detection of a target oligonucleotide in a sample in real time. These methods are compatible with target oligonucleotides amplified using a NEAR reaction.

The present invention features compositions and methods for quantifying detection of a target oligonucleotide in a sample in real time. These methods are compatible with target oligonucleotides amplified using a NEAR reaction.

A method of preparing templates for high throughput nucleic acid sequencing, comprising: a) providing a solid support, wherein said solid support comprises a plurality of nucleic acid primers immobilised adaptor nucleic acid sequences which hybridise to one or more of the nucleic acid primers, and the library preparation having a nucleic acid concentration of 400 pM or less; c) allowing single stranded fragments of template nucleic acid to bind to said nucleic acid primers, thereby immobilising said single stranded fragments on said solid support; and d) repeating steps b) and c) at least three further times, i.e. multiple loadings or rounds of template hybridization to improve occupation rates, e.g., of nanowells on a flow cell.

A method of preparing templates for high throughput nucleic acid sequencing, comprising: a) providing a solid support, wherein said solid support comprises a plurality of nucleic acid primers immobilised adaptor nucleic acid sequences which hybridise to one or more of the nucleic acid primers, and the library preparation having a nucleic acid concentration of 400 pM or less; c) allowing single stranded fragments of template nucleic acid to bind to said nucleic acid primers, thereby immobilising said single stranded fragments on said solid support; and d) repeating steps b) and c) at least three further times, i.e. multiple loadings or rounds of template hybridization to improve occupation rates, e.g., of nanowells on a flow cell.

Embodiments disclosed herein provide methods of using synthetic DNA spike-ins (SDSIs) to detect, prevent, and quantify contamination in amplicon sequencing. These embodiments may, but are not limited to, reveal sample swaps, intra-batch contamination, and, on a larger scale, intra-laboratory contamination. Embodiments disclosed herein also provide synthetic DNA spike-ins for use in amplicon-based sequencing methods.

Embodiments disclosed herein provide methods of using synthetic DNA spike-ins (SDSIs) to detect, prevent, and quantify contamination in amplicon sequencing. These embodiments may, but are not limited to, reveal sample swaps, intra-batch contamination, and, on a larger scale, intra-laboratory contamination. Embodiments disclosed herein also provide synthetic DNA spike-ins for use in amplicon-based sequencing methods.

Provided herein is technology relating to the amplification-based detection of bisulfite-treated DNAs and particularly, but not exclusively, to methods and compositions for multiplex amplification of low-level sample DNA prior to further characterization of the sample DNA. The technology further provides methods for isolating DNA from blood or blood product samples, e.g., plasma samples.