The present disclosure provides methods of sequencing polynucleotides and compounds, compositions for sequencing of polynucleotides, and synthesis of such compositions. The chemical compounds include nucleotides and their analogs which possess a sugar moiety comprising a cleavable chemical group capping the 3′-OH group and a base, but without covalently bounded dye. The cleavable chemical group is reactive to form covalent bond(s) with a dye used to confirm the presence of the expected base-pairing. The cleavable chemical group capping the 3′OH group can be removed together with the covalently bounded dye. Furthermore, after the cleavable chemical group is cleaved, the free 3′-OH group can be active in continued elongation. Example chemical compounds according to the present disclosure are shown as Formulas (II) and (V):

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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 invention provides various orthogonal nucleotide analogues and methods for using combinations of said various orthogonal nucleotide analogues for sequencing by synthesis.

The invention provides various orthogonal nucleotide analogues and methods for using combinations of said various orthogonal nucleotide analogues for sequencing by synthesis.

The present disclosure provides improved nucleic acid sequencing-by-synthesis (SBS) methods, related kits and reagents, and systems for performing such methods using such kits and reagents.

The present disclosure provides improved nucleic acid sequencing-by-synthesis (SBS) methods, related kits and reagents, and systems for performing such methods using such kits and reagents.

The present disclosure provides methods, compositions, and systems employing blocked primers. Aspects of the disclosure include providing a blocked primer reaction mixture that includes a blocked primer and a template nucleic acid component from a single cell; unblocking the blocked primer to produce an active primer reaction mixture and subjecting the activated primer reaction mixture to primer extension conditions, such as nucleic acid implication conditions.

The present disclosure provides methods, compositions, and systems employing blocked primers. Aspects of the disclosure include providing a blocked primer reaction mixture that includes a blocked primer and a template nucleic acid component from a single cell; unblocking the blocked primer to produce an active primer reaction mixture and subjecting the activated primer reaction mixture to primer extension conditions, such as nucleic acid implication conditions.

A polynucleotide sequencing method comprises (i) removing a label and a blocking moiety from a blocked, labeled nucleotide incorporated into a copy polynucleotide strand that is complementary to at least a portion of a template polynucleotide strand; and (ii) washing the removed label and blocking moiety away from the copy strand with a wash solution comprising a first buffer comprising a scavenger compound. Removing the label and blocking moieties may comprise chemically removing the moieties. The first buffer may also comprise an antioxidant and may be used in a scanning buffer used during a nucleotide detection step.