The invention discloses a primer set and a multiplexable library building scheme for constructing an RNA sequencing library with a related reagent kit and an application thereof. The invention also discloses a corresponding data analysis method and a related instrument. The primer set used for RNA sequencing library construction includes the reverse transcription primers containing poly-dT, the 2nd strand cDNA primers particularly contain a random or semi-random portion at their 3′ end for universal initiation of the synthesis at multiple sites, or sequence capturing the 1.sup.st strand of a specific cDNA, as well as their corresponding PCR primer 1 and 2. By this method, the library construction process is simple and the operation is convenient; the time of building the database was significantly shortened; it can carry out a large number of samples in a single run; the analysis process is simpler; the cost of database building, sequencing and analysis is significantly reduced.

The invention discloses a primer set and a multiplexable library building scheme for constructing an RNA sequencing library with a related reagent kit and an application thereof. The invention also discloses a corresponding data analysis method and a related instrument. The primer set used for RNA sequencing library construction includes the reverse transcription primers containing poly-dT, the 2nd strand cDNA primers particularly contain a random or semi-random portion at their 3′ end for universal initiation of the synthesis at multiple sites, or sequence capturing the 1.sup.st strand of a specific cDNA, as well as their corresponding PCR primer 1 and 2. By this method, the library construction process is simple and the operation is convenient; the time of building the database was significantly shortened; it can carry out a large number of samples in a single run; the analysis process is simpler; the cost of database building, sequencing and analysis is significantly reduced.

An improved method for Next Generation Sequencing which relies on the presence of the same distinct unique molecular identifier (UMI) located at each end of a linear nucleic acid molecule so that sequence reads of approximately 2 kb or longer are obtained, and which allows generation of a genomic map without the need of a reference sequence.

An improved method for Next Generation Sequencing which relies on the presence of the same distinct unique molecular identifier (UMI) located at each end of a linear nucleic acid molecule so that sequence reads of approximately 2 kb or longer are obtained, and which allows generation of a genomic map without the need of a reference sequence.

The present invention provides methods to produce a reduced representation of a genome for sequencing and DNA polymorphism detection. In particular, the invention provides PCR-based methods, with normalization of the amplified products using a duplex-specific nuclease, in order to reduce over-representation of PCR products. Oligonucleotides for use in the disclosed method are also provided.

The present invention provides methods to produce a reduced representation of a genome for sequencing and DNA polymorphism detection. In particular, the invention provides PCR-based methods, with normalization of the amplified products using a duplex-specific nuclease, in order to reduce over-representation of PCR products. Oligonucleotides for use in the disclosed method are also provided.

Methods and compositions for maintaining DNA contiguity for sequencing is provided. For example, a plurality of partitions is provided comprising a bead, a forward primer oligonucleotide cleaved from the bead, the forward primer oligonucleotide having a bead-specific barcode and a 3′ end specific for and complementary to a first or second adaptor; a reverse primer oligonucleotide having a 3′ end complementary to the first or second adaptor, wherein the forward primer 3′ end and the reverse primer 3′ end are complementary to different adaptors selected from the first adaptor and the second adaptor; and fragments of genomic DNA reacted with an adapter-loaded tagmentase such that the DNA fragments comprise breakpoints in the fragments and an inserted adaptor at the break points, wherein the tagmentase binds the breakpoints to form linked DNA segments in the form of DNA segment-first adaptor tagmentase second adaptor-(DNA segment-first adaptor tagmentase second adaptor)n-DNA segment, where n is any integer and “-” indicates a covalent linkage.

Methods and compositions for maintaining DNA contiguity for sequencing is provided. For example, a plurality of partitions is provided comprising a bead, a forward primer oligonucleotide cleaved from the bead, the forward primer oligonucleotide having a bead-specific barcode and a 3′ end specific for and complementary to a first or second adaptor; a reverse primer oligonucleotide having a 3′ end complementary to the first or second adaptor, wherein the forward primer 3′ end and the reverse primer 3′ end are complementary to different adaptors selected from the first adaptor and the second adaptor; and fragments of genomic DNA reacted with an adapter-loaded tagmentase such that the DNA fragments comprise breakpoints in the fragments and an inserted adaptor at the break points, wherein the tagmentase binds the breakpoints to form linked DNA segments in the form of DNA segment-first adaptor tagmentase second adaptor-(DNA segment-first adaptor tagmentase second adaptor)n-DNA segment, where n is any integer and “-” indicates a covalent linkage.

Provided herein are methods of amplifying nucleic acids. In particular, methods are provided for amplifying circular RNA molecules. In certain embodiments, circular DNA molecules for amplification are generated from circular RNA molecules. Provided herein are methods for amplifying a nucleic acid. In certain embodiments, a method comprises priming a circular RNA template molecule with one or more DNA primers and extending the primers with a reverse transcriptase to generate a cDNA strand that is a copy of the circular RNA molecule. In certain embodiments, the cDNA strand generated is linear.

Provided herein are methods of amplifying nucleic acids. In particular, methods are provided for amplifying circular RNA molecules. In certain embodiments, circular DNA molecules for amplification are generated from circular RNA molecules. Provided herein are methods for amplifying a nucleic acid. In certain embodiments, a method comprises priming a circular RNA template molecule with one or more DNA primers and extending the primers with a reverse transcriptase to generate a cDNA strand that is a copy of the circular RNA molecule. In certain embodiments, the cDNA strand generated is linear.