Methods of sequencing nucleic acid molecules
Inventors
Oberstrass, Florian • Almogy, Gilad • Lee, Linda • Trepagnier, Eliane • JUNG, Geunwon
Assignees
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Publication Number
US-12674199-B2
Publication Date
2026-07-07
Expiration Date
Abstract
The present disclosure provides methods for nucleic acid sequence identification. The methods may comprise bringing a plurality of nucleic acid molecules in contact with a reaction mixture including a concentration of nucleotides that results in fractional labeling of the nucleic acid molecules. The methods may comprise starting a next reversibly-terminated, sequencing cycle prior to completion of unblocking of reversible terminators in a previous sequencing cycle.
Core Innovation
A method for sequencing is provided by repeatedly performing primer extension reactions on a plurality of nucleic acid molecules that have sequence identity with respect to one another. In each repeating time, a first reaction solution containing a first plurality of nucleotides comprises only one kind of identical purine or pyrimidine nucleotides and includes labeled nucleotides having a first detectable label, and incorporates the first plurality of nucleotides into a first plurality of growing strands of a first subset by a first polymerase in a first primer extension reaction.
After incorporation, nucleotides not incorporated into the first plurality of growing strands are removed by contacting the mixture with a first washing solution. A second reaction solution is then used to incorporate a second plurality of growing strands of a second subset in a second primer extension reaction, where the second plurality comprises the same one kind of identical purine or pyrimidine nucleotides but includes only unlabeled nucleotides at a second concentration higher than the first concentration, followed by removal of non-incorporated nucleotides using a second washing solution.
One or more signals are detected from the first detectable label in the mixture formed after the first washing removal or after the second washing removal, where the signals are indicative that the first plurality of nucleotides is incorporated into the first plurality of growing strands of the first subset. When the repeating is performed for at least four times, the one kind of identical purine or pyrimidine nucleotides used in each time is different, so that sequencing is carried out across multiple repeating cycles using different nucleotide types.
Claims Coverage
The consolidated claim coverage centers on one independent sequencing method that repeats a labeled incorporation and wash step, followed by a higher-concentration unlabeled incorporation and wash step, with signal detection indicative of labeled incorporation. The repeated cycle is performed at least four times and uses a different one kind of identical purine or pyrimidine nucleotide in each time; dependent refinements add label cleaving, additional detectable labels, concentration relationships, and non-overlapping growing strand subsets.
Repeated sequencing cycles with labeled single-type purine or pyrimidine incorporation
Repeating, at least four times, contacting a plurality of nucleic acid molecules with a first reaction solution comprising a first plurality of nucleotides that comprises only one kind of identical purine or pyrimidine nucleotides and comprises labeled nucleotides having a first detectable label, incorporating the first plurality of nucleotides into a first plurality of growing strands of a first subset by a first polymerase in a first primer extension reaction, and using signals from the first detectable label indicative of incorporation.
Wash-and-substitute with higher-concentration unlabeled nucleotides of the same nucleotide type
Subsequent to the labeled incorporation, removing nucleotides not incorporated by contacting the mixture with a first washing solution, then contacting the washed mixture with a second reaction solution comprising a second plurality of nucleotides of the same one kind of identical purine or pyrimidine nucleotides but comprising only unlabeled nucleotides at a second concentration higher than the first concentration, incorporating into a second plurality of growing strands of a second subset, and removing nucleotides not incorporated by contacting with a second washing solution.
Different nucleotide kind across at least four repeating cycles
When the repeating is performed for at least four times, in each time of the at least four times the one kind of identical purine or pyrimidine nucleotides in step (b) is different.
Optional cleaving of the detectable label
After the detecting step, contacting a mixture formed in the detecting step with a cleaving solution to cleave the first detectable label from the first plurality of growing strands of a first subset of the plurality of nucleic acid molecules.
Extension to additional labeled/unlabeled reagent substitutions with a second detectable label
Repeating earlier steps using a third reaction solution with a different labeled nucleotide plurality carrying a second detectable label, then using a fourth reaction solution with only unlabeled nucleotides of that other nucleotide type, and repeating a detection step where signals from the second detectable label indicate incorporation of the third nucleotide plurality.
Quantitative concentration relationship for unlabeled substitution
The second concentration is at least about 10% higher than the first concentration.
Larger quantitative concentration relationship for unlabeled substitution
The second concentration is at least about 500% higher than the first concentration.
Non-overlapping growing strand subsets
The method is characterized by a first plurality of growing strands and a second plurality of growing strands that do not share any common growing strand.
Overall, the independent claim is centered on repeated primer-extension sequencing cycles that use labeled nucleotides of a single identical purine or pyrimidine kind for signal-based incorporation detection, followed by washing and incorporation using higher-concentration unlabeled nucleotides of the same nucleotide kind, with detection performed at least four times using different nucleotide kinds. Dependent claims add refinements such as cleaving the detectable label, adding additional labeled/unlabeled substitutions with a second detectable label, imposing specific concentration relationships, and specifying non-overlapping growing strand sets.
Stated Advantages
Reduced cleaving/washing/scarring and lower cost by using unlabeled nucleotides at higher concentrations (dark polish).
Documented Applications
Sequencing using multi-flow cycles with a signal-calling scheme and example sequencing readout using a three-flow, two-image, single-color method (reported FIG. 3 sequencing example).
Evaluation of phasing behavior and base quality using different flow protocols, including protocols comparing bright polish vs dark polish via labeled vs unlabeled second flows (reported Example 3/4 results and tables).
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