This disclosure relates to methods for increasing capture efficiency of a spatial array using rolling circle amplification of a padlock probe that hybridizes to a capture probe. Also provided are methods for using such spatial arrays to detect a biological analyte in a biological sample.

Ligase mutants of the following (1), (2), or (3):

(1) a ligase mutant comprising an amino acid sequence showing 95% or more identity to the amino acid sequence of SEQ ID NO: 1, and having a nucleic acid-linking activity;

(2) a ligase mutant comprising an amino acid sequence showing 90% or more identity to the amino acid sequence of SEQ ID NO: 2, and having a nucleic acid-linking activity; or

(3) a ligase mutant comprising an amino acid sequence showing 97% or more identity to the amino acid sequence of SEQ ID NO: 3, and having a nucleic acid-linking activity, have excellent properties.

Aspects of the invention include methods for preparing sequencing libraries comprising a plurality of RNA molecules. In some embodiments, the subject methods comprise sequencing at least a portion of a sequencing library to obtain sequencing data or sequence reads from a test sample (e.g., a biological sample from a subject).

A method for producing a single-strand RNA includes a step of causing an RNA ligase which is classified as EC 6.5.1.3 defined by the International Union of Biochemistry and Molecular Biology as an enzyme number and has a nick repair activity to act on a first single-strand RNA and a second single-strand RNA, which have a phosphate group at the 5′ end, and a third single-strand RNA having a hydroxyl group at the 5′ end to link the first single-strand RNA, the second single-strand RNA, and the third single-strand RNA.

A manufacturing method for a single-stranded RNA includes a step of causing an RNA ligase to act on a first single-stranded RNA having a phosphate group at the 5 end and a second single-stranded RNA having a hydroxyl group at the 3 end to link the first single-stranded RNA and the second single-stranded RNA. The first single-stranded RNA is a single-stranded RNA consisting of an X1 region and a Z region in order from the 5 end; the second single-stranded RNA is a single strand consisting of an X2 region, a Y2 region, a Ly linker region, and a Y1 region in order from the 5 end; the X1 region and the X2 region have nucleotide sequences which are complementary to each other, have a length of at least two nucleotides, and have the same number of nucleotides; and the Y1 region and the Y2 region have nucleotide sequences which are complementary to each other, have a length of at least two nucleotides, and have the same number of nucleotides.

A method produces a hairpin single-stranded RNA molecule capable of inhibiting expression of a target gene, the method including: (i) an annealing step of annealing a first single-stranded oligoRNA molecule and a second single-stranded oligoRNA molecule; and (ii) a ligation step of ligating 3 end of the first single-stranded oligoRNA molecule and 5 end of the second single-stranded oligoRNA molecule by an Rnl2 family ligase, wherein a sequence produced by ligating the first single-stranded oligoRNA molecule and the second single-stranded oligoRNA molecule includes a gene expression-inhibiting sequence for the target gene.

The disclosure provides methods, compositions, systems, and kits for the concurrent detection and analysis of different structural and chemical forms of nucleic acids in a sample.

Compositions and methods are provided for ligating polynucleotides having a length that is greater than 8 nucleotides on an RNA splint. The ligation reaction provides consistent results in high or low ATP concentrations. The reaction can occur rapidly and is generally at least 10 fold more efficient than T4DNA ligase under optimal conditions for T4DNA ligase and the reaction time is less than 6 hours for example, less than 1 hour.

Compositions and methods are provided for ligating polynucleotides having a length that is greater than 8 nucleotides on an RNA splint. The ligation reaction provides consistent results in high or low ATP concentrations. The reaction can occur rapidly and is generally at least 10 fold more efficient than T4DNA ligase under optimal conditions for T4DNA ligase and the reaction time is less than 6 hours for example, less than 1 hour.

Modified nucleotides, and methods to modify nucleotides with a moiety or label, such as biotin, that permits their detection and results in a modified nucleotide, and methods of use of the modified nucleotide in quantitative and qualitative assays.