The present disclosure describes technologies that permit sensitive detection of nucleic acids of interest (i.e., nucleic acids whose nucleotide sequence is or includes a target sequence).

A method for sequencing a target polynucleotide includes detecting a first series of nucleotide incorporations complementary to at least a portion of the target polynucleotide. The first series of nucleotide incorporations forms a first complementary polynucleotide. The target nucleotide is secured to a substrate disposed in a sequencing zone of an assembly. The method further includes moving the substrate to which the target nucleotide is secured to a templating zone of the assembly; removing the first complementary polynucleotide when the substrate is disposed at the templating zone of the assembly, the target polynucleotide remaining secured to the substrate; following the removing, moving the substrate to which the target polynucleotide is secured to the sequencing zone; and detecting a second series of nucleotide incorporations complementary to at least a portion of the target polynucleotide, the second series of nucleotide incorporations forming a second complementary polynucleotide.

A method for sequencing a target polynucleotide includes detecting a first series of nucleotide incorporations complementary to at least a portion of the target polynucleotide. The first series of nucleotide incorporations forms a first complementary polynucleotide. The target nucleotide is secured to a substrate disposed in a sequencing zone of an assembly. The method further includes moving the substrate to which the target nucleotide is secured to a templating zone of the assembly; removing the first complementary polynucleotide when the substrate is disposed at the templating zone of the assembly, the target polynucleotide remaining secured to the substrate; following the removing, moving the substrate to which the target polynucleotide is secured to the sequencing zone; and detecting a second series of nucleotide incorporations complementary to at least a portion of the target polynucleotide, the second series of nucleotide incorporations forming a second complementary polynucleotide.

Provided is a PCR-free library construction and sequencing method. A PCR-free high-throughput sequencing method is provided, including the following steps: obtaining a DNA fragment of target size by performing or not performing, based on a size of a nucleic acid sample, fragmentation on the nucleic acid sample; performing end repair and an A-tailing reaction; ligating an adapter containing a barcode; obtaining DNA nanoballs by performing single-strand cyclization and rolling circle replication; and loading and sequencing.

Provided is a PCR-free library construction and sequencing method. A PCR-free high-throughput sequencing method is provided, including the following steps: obtaining a DNA fragment of target size by performing or not performing, based on a size of a nucleic acid sample, fragmentation on the nucleic acid sample; performing end repair and an A-tailing reaction; ligating an adapter containing a barcode; obtaining DNA nanoballs by performing single-strand cyclization and rolling circle replication; and loading and sequencing.

Described herein are methods for rapid, highly multiplexible detection of nucleotides in samples and constructs made to be used in said methods.

Described herein are methods for rapid, highly multiplexible detection of nucleotides in samples and constructs made to be used in said methods.

Disclosed are compositions and methods for template-free nucleic acid synthesis using N-mers and/or anchor primers that comprise at least one XNA or a combination of RNA and DNA.

Disclosed are compositions and methods for template-free nucleic acid synthesis using N-mers and/or anchor primers that comprise at least one XNA or a combination of RNA and DNA.

Polynucleotide sequencing methods include incubating unlabeled nucleotides with a cluster of template polynucleotide strands having the same sequence when the identity of the previously added labeled nucleotide is being detected. The detection step provides time for the addition of the unlabeled nucleotides to be incorporated into the copy strands in which the previously added labeled nucleotide did not get incorporated. Thus, at the end of the detection step, all or most of the copy strands will be in phase and ready to incorporate the appropriate labeled nucleotide in the subsequence incorporate step.