The present disclosure provides compositions and methods pertaining to a next-generation sequencing (NGS) library preparation protocol and method for the optimization of sequencing quality and yield. In particular, the present disclosure provides a novel sequencing platform referred to as OmeSeq, which enables high-fidelity, dosage-sensitive genotyping and strain-level metagenomic profiling of various DNA and RNA templates across animal, plant, microbial, and viral genomes.

The present invention concerns preparation of DNA molecules, such as a library, using a stem-loop oligonucleotide. In particular embodiments, the invention employs a single reaction mixture and conditions. In particular, at least part of the inverted palindrome is removed during the preparation of the molecules to facilitate amplification of the molecules. Thus, in specific embodiments, the DNA molecules are suitable for amplification and are not hindered by the presence of the palindrome.

The present invention concerns preparation of DNA molecules, such as a library, using a stem-loop oligonucleotide. In particular embodiments, the invention employs a single reaction mixture and conditions. In particular, at least part of the inverted palindrome is removed during the preparation of the molecules to facilitate amplification of the molecules. Thus, in specific embodiments, the DNA molecules are suitable for amplification and are not hindered by the presence of the palindrome.

Provided are methods for analyzing properties of a target portion of a polynucleotide using a nanopore. Also provided are methods for nanopore-based analysis of a polynucleotide, the methods comprising modifying a polynucleotide to comprise an enzyme binding site, an enzyme activity blocking structure, and an enzyme displacement region. Such methods further comprise the use of an enzyme that translocates the polynucleotide through a nanopore against a voltage force when the enzyme activity blocking structure is removed. The enzyme blocking structure is reconstituted by movement of the enzyme. Also provided are methods for providing a polynucleotide comprising an abasic region, a region to be analyzed, a GQ fold, and an enzyme binding site, where such methods further comprise removing one enzyme molecule at the abasic region and allowing another enzyme molecule to bind at the enzyme binding site.

Provided are methods for analyzing properties of a target portion of a polynucleotide using a nanopore. Also provided are methods for nanopore-based analysis of a polynucleotide, the methods comprising modifying a polynucleotide to comprise an enzyme binding site, an enzyme activity blocking structure, and an enzyme displacement region. Such methods further comprise the use of an enzyme that translocates the polynucleotide through a nanopore against a voltage force when the enzyme activity blocking structure is removed. The enzyme blocking structure is reconstituted by movement of the enzyme. Also provided are methods for providing a polynucleotide comprising an abasic region, a region to be analyzed, a GQ fold, and an enzyme binding site, where such methods further comprise removing one enzyme molecule at the abasic region and allowing another enzyme molecule to bind at the enzyme binding site.

The present invention relates to a method of using nanopores to obtain sequence information of sample DNAs in ss test DNAs. The method comprises using speed bumps to stall the ss test DNAs in the nanopores at random positions of the ss test DNAs to obtain sequence information of each and every nucleotides of the sample DNAs, and to construct the whole sequences of the sample DNAs. The present invention also relates to identification and/or isolation of test DNAs having desired sequence(s) using nanopore detectors facilitated by speed bump.

The present invention relates to a method of using nanopores to obtain sequence information of sample DNAs in ss test DNAs. The method comprises using speed bumps to stall the ss test DNAs in the nanopores at random positions of the ss test DNAs to obtain sequence information of each and every nucleotides of the sample DNAs, and to construct the whole sequences of the sample DNAs. The present invention also relates to identification and/or isolation of test DNAs having desired sequence(s) using nanopore detectors facilitated by speed bump.

The present disclosure provides polynucleotide-based inhibitors for reversible activation of DNA polymerases. Use of lower Tm polynucleotide-based inhibitors allow PCR reaction assembly at room temperature while activating polymerase at higher PCR primer annealing temperatures, where the reversible nature of the inhibition additionally improves priming specificity during each PCR cycle. Additionally, temperature controlled inactivation of polymerase activity after PCR or other polymerase based enzymatic incubation eliminates a purification step when needed for compatibility with subsequent enzymatic incubations. For this application, the T.sub.m of the polynucleotide-based inhibitor is higher than the desired reaction conditions of the subsequent enzymatic incubation.

The present disclosure provides polynucleotide-based inhibitors for reversible activation of DNA polymerases. Use of lower Tm polynucleotide-based inhibitors allow PCR reaction assembly at room temperature while activating polymerase at higher PCR primer annealing temperatures, where the reversible nature of the inhibition additionally improves priming specificity during each PCR cycle. Additionally, temperature controlled inactivation of polymerase activity after PCR or other polymerase based enzymatic incubation eliminates a purification step when needed for compatibility with subsequent enzymatic incubations. For this application, the T.sub.m of the polynucleotide-based inhibitor is higher than the desired reaction conditions of the subsequent enzymatic incubation.

Disclosed herein is a method of covalently crosslinking DNA strands. In certain aspects, the method comprises incubating a hybridized, double-stranded DNA polynucleotide (dsDNA), comprising a probe strand that comprises an abasic (Ap) residue and an at least partially complementary target strand that comprises a 2′-deoxyadenosine (dA) residue, wherein incubation occurs under conditions that allow for a covalent crosslinking reaction to occur between the Ap residue in the probe strand and the dA residue in the target strand.