The present invention provides methods, compositions, and systems for distributing molecules and complexes into reaction sites. In particular, the methods, compositions, and systems of the present invention result in loading of polymerase enzyme complexes into a predetermined number of reaction sites, including nanoscale wells.

The present invention relates to a method for introducing mutations into at least one target nucleic acid molecule comprising (a) providing at least one sample comprising at least one target nucleic acid molecule; and (b) amplifying the at least one target nucleic acid molecule using a low bias DNA polymerase. The present further relates to a use of a low bias DNA polymerase in a method for introducing mutations into one or more nucleic acid molecule(s), a group of sample tags, a method for designing the group of sample tags, a computer readable medium, and a method for preferentially amplifying target nucleic acid molecules.

Disclosed herein include systems, methods, compositions, and kits for molecular barcoding on the 5′-end of a nucleic acid target. After barcoding a nucleic acid target using an oligonucleotide barcode comprising a target binding region and a molecular label to generate a barcoded nucleic acid molecule, an oligonucleotide comprising a complement of the target binding region can be added to generate a barcoded nucleic acid molecule comprising the target-binding region and the complement of the target-binding region. A stem loop is formed with intra-molecular hybridization of the barcoded nucleic acid molecule, which can be extended to generate an extended barcoded nucleic acid molecule comprising the molecular label and a complement of the molecular label.

Disclosed herein include systems, methods, compositions, and kits for molecular barcoding on the 5′-end of a nucleic acid target. After barcoding a nucleic acid target using an oligonucleotide barcode comprising a target binding region and a molecular label to generate a barcoded nucleic acid molecule, an oligonucleotide comprising a complement of the target binding region can be added to generate a barcoded nucleic acid molecule comprising the target-binding region and the complement of the target-binding region. A stem loop is formed with intra-molecular hybridization of the barcoded nucleic acid molecule, which can be extended to generate an extended barcoded nucleic acid molecule comprising the molecular label and a complement of the molecular label.

Provided are sequencing-by-binding methods of detecting cognate nucleotides using a crippled DNA polymerizing enzyme that possesses the ability to bind the next correct nucleotide downstream of a primer in a template-dependent fashion, but does not possess the activity needed to promote phosphodiester bond formation. Use of the crippled DNA polymerase permits interrogation of one nucleotide at a time, without incorporation of any nucleotide. Labeled nucleotides, such as fluorescently labeled nucleotides, can be used in conjunction with the crippled DNA polymerase to establish cognate nucleotide identity in a rapid manner.

Provided are sequencing-by-binding methods of detecting cognate nucleotides using a crippled DNA polymerizing enzyme that possesses the ability to bind the next correct nucleotide downstream of a primer in a template-dependent fashion, but does not possess the activity needed to promote phosphodiester bond formation. Use of the crippled DNA polymerase permits interrogation of one nucleotide at a time, without incorporation of any nucleotide. Labeled nucleotides, such as fluorescently labeled nucleotides, can be used in conjunction with the crippled DNA polymerase to establish cognate nucleotide identity in a rapid manner.

Provided are methods and systems for detecting formation of nucleotide-specific ternary complexes comprising a DNA polymerase, a nucleic acid, and a nucleotide complementary to the templated base of the primed template nucleic acid. The methods and systems facilitate determination of the next correct nucleotide without requiring chemical incorporation of the nucleotide into the primer. These results can even be achieved in procedures employing unlabeled, native nucleotides.

Provided are methods and systems for detecting formation of nucleotide-specific ternary complexes comprising a DNA polymerase, a nucleic acid, and a nucleotide complementary to the templated base of the primed template nucleic acid. The methods and systems facilitate determination of the next correct nucleotide without requiring chemical incorporation of the nucleotide into the primer. These results can even be achieved in procedures employing unlabeled, native nucleotides.

Methods of determining polymerase fidelity are provided. In one embodiment, the method comprises filling a gapped plasmid with a polymerase to form a gap-filled plasmid, wherein the gap-filled plasmid comprises a gene encoding an protein that is functional or non-functional depending on the polymerase fidelity; forming a plurality of partitions from a solution comprising the gap-filled plasmid and a label for detecting the presence of the plasmid; detecting the presence of the gap-filled plasmid in one or more of the partitions; and determining the fidelity of the polymerase by determining a ratio of partitions containing the gene encoding a functional protein to partitions containing a gene encoding a non-functional protein.

Methods of determining polymerase fidelity are provided. In one embodiment, the method comprises filling a gapped plasmid with a polymerase to form a gap-filled plasmid, wherein the gap-filled plasmid comprises a gene encoding an protein that is functional or non-functional depending on the polymerase fidelity; forming a plurality of partitions from a solution comprising the gap-filled plasmid and a label for detecting the presence of the plasmid; detecting the presence of the gap-filled plasmid in one or more of the partitions; and determining the fidelity of the polymerase by determining a ratio of partitions containing the gene encoding a functional protein to partitions containing a gene encoding a non-functional protein.