Omniome, Inc.

Pacific Biosciences of California, Inc.

Omnionie, Inc.

Pinar Iyidogan

Kandaswamy Vijayan

Mark C Wallen

Ying Lin Liu

Arnold Oliphant

Brittany Ann Rohrman

Fabian Block

Morassa Mohseni Middleton

Michael James Schmidt

Bridget Kidd

Mariam Iftikhar

Lewis Churchfield

Methods, compositions, kits and apparatuses that include a fluid, the fluid containing a ternary complex and Li, wherein the ternary complex includes a primed template nucleic acid, a polymerase, and a nucleotide cognate for the next correct base for the primed template nucleic acid molecule. As an alternative or addition to Li, the fluid can contain betaine or a metal ion that inhibits polymerase catalysis such as Ca. In addition to Li, the fluid can contain polyethylenimine (PEI) with or without betaine.

Methods and compositions for stabilizing nucleic acid-nucleotide-polymerase complexes

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.

Method of nucleic acid sequence determination

Provided are nucleic acids encoding engineered polymerases comprising at least one modification in a motif A and/or at least one modification in a motif B of the polymerase and engineered polymerases encoded by the nucleic acids. Also provided are engineered DNA polymerases comprising a variant of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, the variant being at least 80% identical to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 and comprising an amino acid substitution at one or more positions selected from the group consisting of L408, Y409, P410, R484, A/L485, and I486. Methods, vectors, kits, and compositions comprising the nucleic acids and compositions, methods and kits comprising the engineered polymerases are also provided.

Engineered polymerases for improved sequencing

Provided herein are engineered DNA polymerases comprising modifications improving accuracy and processivity of the polymerase including modifications in the Motif A region, optionally, along with additional modifications in the palm and/or exonuclease domains of the polymerase. Also provided are nucleic acids encoding the engineered DNA polymerases comprising modifications in motif A of the polymerase, optionally, with additional modifications. Methods, vectors, kits, and compositions comprising the nucleic acids and compositions, methods and kits comprising the engineered polymerases are also provided.

Engineered polymerases for improved sequencing by binding

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.

Sequencing method employing ternary complex destabilization to identify cognate nucleotides

Provided are methods and systems for reducing the time needed for sequencing nucleic acids. The approach relies on detecting formation of nucleotide-specific ternary complexes comprising a polymerase (e.g., a DNA polymerizing enzyme), a primed template nucleic acid molecule, 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, as well as the subsequent next correct nucleotide from a cycle of examining four different nucleotides without requiring chemical incorporation of any nucleotide into the primer.

Sequencing method for rapid identification and processing of cognate nucleotide pairs

Provided are engineered DNA polymerases exhibiting modified functionality, and polynucleotides encoding same. Modified features include: (1) reduced catalytic activity in the presence of magnesium ions and/or (2) reduced affinity for primed template nucleic acid molecules in the absence of cognate nucleotide, and an ability to discriminate between cognate and non-cognate nucleotides under low salt conditions. Sequencing By Binding&#x2122; procedures employing the engineered polymerases have certain advantages. The engineered polymerases can have other uses as well.

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Pinar Iyidogan