Provided includes methods, compositions and systems for single molecule seeding and amplification on a flow cell. In some embodiments, nucleic acids are isothermally seeded and amplified on a flow cell comprising multiple binding areas (e.g., pads), resulting in an ensemble of substantially the same amplified molecules on each of the binding areas.

The present invention relates to a method for the highly specific, targeted capture of regions of human genomes and transcriptomes from the blood, i.e. from cell free circulating DNA, exosomes, microRNA, circulating tumor cells, or total blood cells, to allow for the highly sensitive detection of mutation, expression, copy number, translocation, alternative splicing, and methylation changes using combined nuclease, ligation, polymerase, and massively parallel sequencing reactions. The method generates a collection of different circular chimeric single-stranded nucleic acid constructs, suitable for sequencing on multiple platforms. In some embodiments, each construct of the collection comprised a first single stranded segment of original genomic DNA from a host organism and a second single stranded synthetic nucleic acid segment that is linked to the first single stranded segment and comprises a nucleotide sequence that is exogenous to the host organism. These chimeric constructs are suitable for identifying and enumerating mutations, copy changes, translocations, and methylation changes. In other embodiments, input mRNA, lncRNA, or miRNA is used to generate circular DNA products that reflect the presence and copy number of specific mRNA's, lncRNA's splice-site variants, translocations, and miRNA.

The present invention relates to a method for the highly specific, targeted capture of regions of human genomes and transcriptomes from the blood, i.e. from cell free circulating DNA, exosomes, microRNA, circulating tumor cells, or total blood cells, to allow for the highly sensitive detection of mutation, expression, copy number, translocation, alternative splicing, and methylation changes using combined nuclease, ligation, polymerase, and massively parallel sequencing reactions. The method generates a collection of different circular chimeric single-stranded nucleic acid constructs, suitable for sequencing on multiple platforms. In some embodiments, each construct of the collection comprised a first single stranded segment of original genomic DNA from a host organism and a second single stranded synthetic nucleic acid segment that is linked to the first single stranded segment and comprises a nucleotide sequence that is exogenous to the host organism. These chimeric constructs are suitable for identifying and enumerating mutations, copy changes, translocations, and methylation changes. In other embodiments, input mRNA, lncRNA, or miRNA is used to generate circular DNA products that reflect the presence and copy number of specific mRNA's, lncRNA's splice-site variants, translocations, and miRNA.

The present disclosure relates in some aspects to methods and compositions for analysis of a target nucleic acid, such as in situ detection of a region of interest in a polynucleotide in a tissue sample. In some embodiments, provided herein are templated ligation probes (e.g., RNA-templated ligation probes) and selector probes for generation of a circularized ligated probe comprising an insertion sequence of a selector probe, wherein the circularized ligated probe is amplified in a rolling circle amplification reaction to generate a product that is detected in the sample.

The present disclosure relates in some aspects to methods and compositions for analysis of a target nucleic acid, such as in situ detection of a region of interest in a polynucleotide in a tissue sample. In some embodiments, provided herein are templated ligation probes (e.g., RNA-templated ligation probes) and selector probes for generation of a circularized ligated probe comprising an insertion sequence of a selector probe, wherein the circularized ligated probe is amplified in a rolling circle amplification reaction to generate a product that is detected in the sample.

A method for determining sequences from sense and antisense strands of a nucleic acid, including (a) providing a nucleic acid cluster attached to a solid support, wherein the nucleic acid cluster includes a sense strand and an antisense strand of a concatemer, the concatemer including multiple copies of a sequence unit, the sequence unit including a target sequence and a primer binding site; (b) hybridizing a primer to a primer binding site in the antisense strand; (c) extending the primer along the antisense strand to determine the sequence from at least a portion of the target sequence in the antisense strand; (d) hybridizing a second primer to a primer binding site in the sense strand; and (e) extending the second primer along the sense strand to determine the sequence from at least a portion of the target sequence in the sense strand.

A method for determining sequences from sense and antisense strands of a nucleic acid, including (a) providing a nucleic acid cluster attached to a solid support, wherein the nucleic acid cluster includes a sense strand and an antisense strand of a concatemer, the concatemer including multiple copies of a sequence unit, the sequence unit including a target sequence and a primer binding site; (b) hybridizing a primer to a primer binding site in the antisense strand; (c) extending the primer along the antisense strand to determine the sequence from at least a portion of the target sequence in the antisense strand; (d) hybridizing a second primer to a primer binding site in the sense strand; and (e) extending the second primer along the sense strand to determine the sequence from at least a portion of the target sequence in the sense strand.

The subject invention pertains to methods for the analysis of pooled samples without the need of retesting through the use of oligonucleotide hybridization and target-specific amplification reactions. Specifically, a series of identifier oligonucleotides with different sequence compositions, each corresponding to a distinct sample, are combined into the target template of interest through nucleic acid synthesis. The aforementioned products are pooled together, and the pooled samples are amplified and detected using the probe-based hybridization assay or a size separation module to identify if any of the pool of samples test positive, as well as simultaneously identifying which sample is positive for the targeted sequence.

The subject invention pertains to methods for the analysis of pooled samples without the need of retesting through the use of oligonucleotide hybridization and target-specific amplification reactions. Specifically, a series of identifier oligonucleotides with different sequence compositions, each corresponding to a distinct sample, are combined into the target template of interest through nucleic acid synthesis. The aforementioned products are pooled together, and the pooled samples are amplified and detected using the probe-based hybridization assay or a size separation module to identify if any of the pool of samples test positive, as well as simultaneously identifying which sample is positive for the targeted sequence.

Disclosed is a method for determining a nucleotide sequence of a target nucleic acid. The method comprises: providing a pool of amplicons; sequencing each amplicon in the pool of amplicons to obtain sequence information of each amplicon; comparing a part of the sequence information of each amplicon with at least a part of the sequence of the target specific primer section, wherein the part of the sequence information of each amplicon is a sequence starting from position X-y and y is positive integer; determining whether the part of the sequence information of each amplicon comprises at least the part of the sequence of the target specific primer section; and determining accurate sequence of the target region using sequence information which comprises at least the part of the sequence of the target-specific primer section.