Generalized stochastic super-resolution sequencing

A method for sequencing polynucleotides comprising:

• attaching a first DNA template molecule to a first attachment element of a plurality of attachment elements of a sample container and attaching a second DNA template molecule to a second attachment element of the plurality of attachment elements, wherein a distance between the first attachment element and the second attachment element is less than Abbe's limit;
• providing a stochastic photo-switching chemistry to the sample container, the stochastic photo-switching chemistry comprising a set of nucleotides that have a 5′ diphosphate quencher molecule and 3′ phosphate block with a label moiety, a polymerase to incorporate a nucleotide of the set of nucleotides and cleave the 5′ diphosphate quencher molecule, and an enzyme to cleave the 3′ phosphate block with the label moiety;
• imaging a series of on and off events for the first DNA template molecule in real-time as the on and off events are occurring, each on event occurring when the polymerase incorporates a nucleotide of the set of nucleotides and cleaves the 5′ diphosphate quencher molecule and each off event occurring when the enzyme cleaves the 3′ phosphate block with the label moiety;
• imaging a series of on and off events for the second DNA template molecule in real-time as the on and off events are occurring, wherein the series of on and off events for the first DNA template molecule and the series of on and off events for the second DNA template molecule are stochastic and not synchronized;
• and
• controlling a rate at which the on and off events occur;
• wherein controlling the rate at which the on and off events occur comprises adjusting concentrations of nucleotides and enzymes used in the stochastic photo-switching chemistry so that a probability that an on event for a nucleotide base for the first DNA template molecule will occur at the same time as an on event for a nucleotide base for the second DNA template molecule is lower than a determined error rate in a sequencing application in which the method is applied.