Described herein are methods for determining a sequence of a region of interest from an mRNA molecule. Sequenced polynucleotides can include a barcode region, a homopolymer region (e.g., a poly-A region), and a target region associated with the mRNA molecule. According to some methods, the barcode region omits the same base present in the homopolymer region. According to some methods, extension of the primer used for sequencing is stalled within the homopolymer region. According to some methods, sequencing flow cycles and the different barcode regions of the polynucleotides configured are such that the primer is extended to the end of the barcode region across the plurality of polynucleotides before being extended into the homopolymer region. According to some methods, two primers or a cleavable primer is used to separately sequence the barcode region and the target region.

Described herein are methods for determining a sequence of a region of interest from an mRNA molecule. Sequenced polynucleotides can include a barcode region, a homopolymer region (e.g., a poly-A region), and a target region associated with the mRNA molecule. According to some methods, the barcode region omits the same base present in the homopolymer region. According to some methods, extension of the primer used for sequencing is stalled within the homopolymer region. According to some methods, sequencing flow cycles and the different barcode regions of the polynucleotides configured are such that the primer is extended to the end of the barcode region across the plurality of polynucleotides before being extended into the homopolymer region. According to some methods, two primers or a cleavable primer is used to separately sequence the barcode region and the target region.

Provided herein, in some aspects, are methods of imaging molecules without a microscope or other specialized equipment, referred to herein as “microscope-free imaging (MFI).” Herein, “molecular instruments” (e.g., DNA-based and protein-based molecules) are used, instead of microscopes, in a “bottom-up” approach for inspecting molecular targets.

Provided herein, in some aspects, are methods of imaging molecules without a microscope or other specialized equipment, referred to herein as “microscope-free imaging (MFI).” Herein, “molecular instruments” (e.g., DNA-based and protein-based molecules) are used, instead of microscopes, in a “bottom-up” approach for inspecting molecular targets.

The present invention provides methods and systems for sequencing long nucleic acid fragment. The present invention also provides a method of sequencing a target polynucleotide with fewer probes. Further, the present invention provides a method of sequencing a target polynucleotide with longer reads. Locus-specific, ligation-assisted sequencing/genotyping method and ligation-captured sequencing method are also provided in the present invention. The methods of the present invention allow low-cost, high-throughput and accurate sequencing of nucleic acids.

Methods of coupling adaptors to a target nucleic acid include coupling a first adaptor to a first end of the target nucleic acid to form a coupled first adaptor. A portion of a second adaptor is hybridized to a portion of the coupled first adaptor to form a hybridized second adaptor having a single-stranded 3′-end. The hybridized second adaptor is coupled to a second end of the target nucleic acid to form an adaptor-flanked product having at least a part of the first adaptor coupled to the first end of the target nucleic acid and at least a part of the second adaptor coupled to the second end of the target nucleic acid. These methods can minimize the formation of adaptor-dimers that may be problematic in subsequent complementary nucleic acid strand synthesis, amplification, and sequencing.

Methods of coupling adaptors to a target nucleic acid include coupling a first adaptor to a first end of the target nucleic acid to form a coupled first adaptor. A portion of a second adaptor is hybridized to a portion of the coupled first adaptor to form a hybridized second adaptor having a single-stranded 3′-end. The hybridized second adaptor is coupled to a second end of the target nucleic acid to form an adaptor-flanked product having at least a part of the first adaptor coupled to the first end of the target nucleic acid and at least a part of the second adaptor coupled to the second end of the target nucleic acid. These methods can minimize the formation of adaptor-dimers that may be problematic in subsequent complementary nucleic acid strand synthesis, amplification, and sequencing.

An object of the present invention is to provide a novel method for designing a primer ensuring reactivity and discriminatory power in a method for detecting a single base substitution based on an ASP-PCR method and to provide a method for easily detecting multiple point mutations within overlapping amplicons, particularly, two adjacent single base substitutions. The single base substitutions can easily be detected by using a mutant primer in which the base of the third nucleotide from the 3′ end corresponds to the base of a mutant nucleotide of a single base substitution contained in a nucleic acid sample, in which the base of the second nucleotide from the 3′ end is not complementary to the base of the corresponding nucleotide of the nucleic acid, and in which the bases of the other nucleotides are complementary to the bases of the corresponding nucleotides of the nucleic acid.

An object of the present invention is to provide a novel method for designing a primer ensuring reactivity and discriminatory power in a method for detecting a single base substitution based on an ASP-PCR method and to provide a method for easily detecting multiple point mutations within overlapping amplicons, particularly, two adjacent single base substitutions. The single base substitutions can easily be detected by using a mutant primer in which the base of the third nucleotide from the 3′ end corresponds to the base of a mutant nucleotide of a single base substitution contained in a nucleic acid sample, in which the base of the second nucleotide from the 3′ end is not complementary to the base of the corresponding nucleotide of the nucleic acid, and in which the bases of the other nucleotides are complementary to the bases of the corresponding nucleotides of the nucleic acid.

The invention provides methods, compositions, kits and devices for the detection of target molecules. In some embodiments, the invention allows for multiplexed target molecule detection.