Provided are methods for preparation and analysis of nucleic acids. Some embodiments include reverse transcribing the RNA with barcoded primers to produce cDNA while maintaining the DNA in the sample, sequencing the DNA and cDNA together, and differentiating the sequenced DNA and cDNA using the barcode or barcodes of the primers. Some embodiments include analyzing the DNA and cDNA sequences of multiple samples separating reads into k-mers, and comparing the k-mers between samples to identify differential sequences between the sequences of the samples.

Provided are methods for preparation and analysis of nucleic acids. Some embodiments include reverse transcribing the RNA with barcoded primers to produce cDNA while maintaining the DNA in the sample, sequencing the DNA and cDNA together, and differentiating the sequenced DNA and cDNA using the barcode or barcodes of the primers. Some embodiments include analyzing the DNA and cDNA sequences of multiple samples separating reads into k-mers, and comparing the k-mers between samples to identify differential sequences between the sequences of the samples.

Provided herein is a polymerase-free enzyme mix (FRAG) for fragmenting double-stranded DNA. In some embodiments the enzyme mix may comprise a double-stranded DNA nickase and at least one of a DNA ligase capable of sealing a nick within a DNA, and a single-strand specific DNA nuclease. Methods for fragmenting double-stranded DNA are also provided.

Provided herein is a polymerase-free enzyme mix (FRAG) for fragmenting double-stranded DNA. In some embodiments the enzyme mix may comprise a double-stranded DNA nickase and at least one of a DNA ligase capable of sealing a nick within a DNA, and a single-strand specific DNA nuclease. Methods for fragmenting double-stranded DNA are also provided.

Provided is a preparation method for a DNA library, comprising a pre-library preparation process, the pre-library preparation process comprising DNA preparation, end repair and 3′ A-tailing, linker connection using an anti-contamination linker, linker connected product purification, pre-library amplification, and amplified pre-library purification. Also provided are a use of the anti-contamination linker in preparing a test kit for DNA library capture, and a method for performing bioinformatic analysis on the DNA library prepared by means of the preparation method of the present invention. The preparation method of the present invention reduces the risk of cross-contamination between samples.

Provided is a preparation method for a DNA library, comprising a pre-library preparation process, the pre-library preparation process comprising DNA preparation, end repair and 3′ A-tailing, linker connection using an anti-contamination linker, linker connected product purification, pre-library amplification, and amplified pre-library purification. Also provided are a use of the anti-contamination linker in preparing a test kit for DNA library capture, and a method for performing bioinformatic analysis on the DNA library prepared by means of the preparation method of the present invention. The preparation method of the present invention reduces the risk of cross-contamination between samples.

The present invention relates to a method for generating a library of different polynucleotide molecules, by ligating a double-stranded polynucleotide to a plurality of different target polynucleotide duplexes, the double-stranded polynucleotide comprising: (a) a first strand comprising an annealed portion and an overhang portion; and (b) a second strand consisting essentially of an annealed portion, wherein the second strand is complementary to and annealed to the annealed portion of the first strand.

The present invention relates to a method for generating a library of different polynucleotide molecules, by ligating a double-stranded polynucleotide to a plurality of different target polynucleotide duplexes, the double-stranded polynucleotide comprising: (a) a first strand comprising an annealed portion and an overhang portion; and (b) a second strand consisting essentially of an annealed portion, wherein the second strand is complementary to and annealed to the annealed portion of the first strand.

A method includes (i) providing an RNA polynucleotide; (ii) modifying the RNA polynucleotide by annealing and ligating a polynucleotide comprising a 3′ terminal random multimer segment and having a stem-loop form; (iii) optionally performing a reverse transcription of the RNA polynucleotide; (iv) cleaving the stem-loop segment of the annealed polynucleotide to yield a 3′ A overhang; (v) connecting an adaptor polynucleotide complex associated with an RNA translocase enzyme and at least one cholesterol tether segment to the polynucleotide obtained in step (iv); (vi) contacting the modified RNA polynucleotide obtained in step (v) with a transmembrane pore such that the RNA translocase controls the movement of the RNA polynucleotide through the transmembrane pore and the cholesterol tether anchors the RNA polynucleotide in the vicinity of the transmembrane pore; and (vii) taking one or more measurements during the movement of the RNA polynucleotide through the transmembrane pore Other features are also disclosed.

A method includes (i) providing an RNA polynucleotide; (ii) modifying the RNA polynucleotide by annealing and ligating a polynucleotide comprising a 3′ terminal random multimer segment and having a stem-loop form; (iii) optionally performing a reverse transcription of the RNA polynucleotide; (iv) cleaving the stem-loop segment of the annealed polynucleotide to yield a 3′ A overhang; (v) connecting an adaptor polynucleotide complex associated with an RNA translocase enzyme and at least one cholesterol tether segment to the polynucleotide obtained in step (iv); (vi) contacting the modified RNA polynucleotide obtained in step (v) with a transmembrane pore such that the RNA translocase controls the movement of the RNA polynucleotide through the transmembrane pore and the cholesterol tether anchors the RNA polynucleotide in the vicinity of the transmembrane pore; and (vii) taking one or more measurements during the movement of the RNA polynucleotide through the transmembrane pore Other features are also disclosed.