Provided herein are engineered variants of archaeal, prokaryotic, and eukaryotic polymerases that exhibit enhanced thermostability, enhanced incorporation of 3′ modified nucleotides, and improved uracil-tolerance, in polymerase-catalyzed nucleotide extension reactions relative to wild type polymerase enzymes. Also provided are uses of the engineered polymerases for forming complexed polymerases, forming binding complexes and forming ternary complexes, and uses for conducting nucleic acid sequencing reactions.

The present disclosure provides a method for assembly of genomic DNA using multiplex end-tagging amplification of genomic fragments.

The present disclosure provides a method for assembly of genomic DNA using multiplex end-tagging amplification of genomic fragments.

Disclosed are methods for isolating polymerase complexes from a mixture of polymerase complex components. The polymerase complexes can comprise a nanopore to provide isolated nanopore sequencing complexes. The methods relate to the positive and negative isolation of the polymerase complexes and/or nanopore sequencing complexes. Also disclosed is a nucleic acid adaptor for isolating active polymerase complexes, polymerase complexes comprising the nucleic acid adaptor, and methods for isolating active polymerase complexes using the nucleic acid adaptor.

Disclosed are methods for isolating polymerase complexes from a mixture of polymerase complex components. The polymerase complexes can comprise a nanopore to provide isolated nanopore sequencing complexes. The methods relate to the positive and negative isolation of the polymerase complexes and/or nanopore sequencing complexes. Also disclosed is a nucleic acid adaptor for isolating active polymerase complexes, polymerase complexes comprising the nucleic acid adaptor, and methods for isolating active polymerase complexes using the nucleic acid adaptor.

Provided herein is a method of sequencing a target double stranded nucleic acid. The method comprises contacting the double stranded nucleic acid with a reagent as described herein to form a construct and sequencing the construct using a single-molecule sequencing technique as described herein. Associated products and kits are further provided.

Provided herein is a method of sequencing a target double stranded nucleic acid. The method comprises contacting the double stranded nucleic acid with a reagent as described herein to form a construct and sequencing the construct using a single-molecule sequencing technique as described herein. Associated products and kits are further provided.

A method for purifying nucleotides is provided, that includes preparing a solution comprising (a) 3′-blocked nucleotides, (b) 3′-OH nucleotides, (c) a polishing polymerase, and (d) a template. The polishing polymerase and the template are used to selectively polymerize the 3′-OH nucleotides and thus reduce a concentration in the solution of the 3′-OH nucleotides relative to the 3′-blocked nucleotides.

A method for purifying nucleotides is provided, that includes preparing a solution comprising (a) 3′-blocked nucleotides, (b) 3′-OH nucleotides, (c) a polishing polymerase, and (d) a template. The polishing polymerase and the template are used to selectively polymerize the 3′-OH nucleotides and thus reduce a concentration in the solution of the 3′-OH nucleotides relative to the 3′-blocked nucleotides.

The disclosure provides methods for processing nucleic acid populations containing different forms (e.g., RNA and DNA, single-stranded or double-stranded) and/or extents of modification (e.g., cytosine methylation, association with proteins). These methods accommodate multiple forms and/or modifications of nucleic acid in a sample, such that sequence information can be obtained for multiple forms. The methods also preserve the identity of multiple forms or modified states through processing and analysis, such that analysis of sequence can be combined with epigenetic analysis.