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US 12,385,160 B2Granted
Enzyme screening methods
AssigneesRoche Sequencing Solutions, Inc., President and Fellows of Harvard College
InventorsAruna Ayer, George M. Church, Mirko Palla, Francois Pepin, Sukanya Srinivasa Rao Arun Punthambaker, Peter B. Stranges
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Abstract
The present disclosure is directed to compositions and methods for deriving a plurality of kinetics parameters (240) for at least two different enzyme variants in a multiplex manner using nanopore-based sequencing. In some embodiments, the systems and methods may be used to screen different nanopore variants, or different combinations of both nanopore variants and enzyme variants.
Claims14
A method of screening a plurality of different enzyme variants using nanopore-based sequencing comprising:
- obtaining a biochip including a plurality of different nanopore sequencing complexes, wherein each different nanopore sequencing complex of the plurality of different nanopore sequencing complexes includes a polynucleotide having a unique molecular barcode, and wherein at least two of the plurality of different nanopore sequencing complexes have different enzyme variants, wherein the plurality of different enzyme variants are polymerase variants or reverse transcriptase variants;
- generating a sequencing data set for each different nanopore sequencing complex loaded onto the biochip;
- classifying each of the generated sequencing data sets as associated with one different enzyme variant of the plurality of different enzyme variants based on identifications of the unique molecular barcodes included in the polynucleotides of the different nanopore sequencing complexes;
- and
- deriving a plurality of parameters for each one of the enzyme variants of the plurality of different enzyme variants, wherein the plurality of parameters for each one of the different enzyme variants are derived based on the classified sequence data sets associated with the respective one of the different enzyme variants.
2 more elements
A method of screening at least two enzyme variants using nanopore-base sequencing comprising:
- obtaining a biochip including a plurality of individually addressable nanopore sequencing complexes, and wherein the obtained biochip comprises at least first and second different nanopore sequencing complexes, the first nanopore sequencing complex comprising a first enzyme variant and a first polynucleotide, and the second nanopore sequencing complex comprising a second enzyme variant and a second polynucleotide, wherein the first and second polynucleotides each include a different molecular barcode, and wherein the first and second enzyme variants are different;
- generating sequencing data sets for at least each of the first and second nanopore sequencing complexes, wherein the generated sequencing data sets for the at least the first and second nanopore sequencing complexes are derived from signals detected by a sensing circuit associated with the at least the first and second nanopore sequencing complexes;
- classifying each of the generated sequencing data sets as associated with at least either the first enzyme variant or the second enzyme variant, wherein the sequence data sets are each classified as associated with the at least either the first enzyme variant or the second enzyme variant based on identifications of at least the unique molecular barcodes included with the first and second polynucleotides;
- and
- deriving a plurality of kinetics parameters for each of the first and second enzyme variants based on the classified data sets associated with the first enzyme variant or the second enzyme variant,
- wherein the at least two enzyme variants are polymerase variants or reverse transcriptase variants.
3 more elements
A method of characterizing a plurality of different nanopore variants using nanopore-based sequencing comprising:
- a. obtaining a biochip including a plurality of different nanopore sequencing complexes wherein each different nanopore sequencing complex of the plurality of different nanopore sequencing complexes are individually addressable, wherein each different nanopore sequencing complex is associated with a different polynucleotide, wherein each different polynucleotide includes a unique molecular barcode and the same common region, and wherein at least two of the different nanopore sequencing complexes of the plurality of different nanopore sequencing complexes have different nanopore variants;
- b. generating a sequencing data set for each of the plurality of different nanopore sequencing complexes by sequencing each of the different polynucleotides associated with each of the plurality of different nanopore sequencing complexes, wherein each of the generated sequencing data sets is derived from signals detected by a sensing circuit associated with each of the individually addressable nanopore sequencing complexes;
- c. associating each of the generated sequencing data sets with one of the different nanopore variants based on identifications of the different unique molecular barcodes included in each of the different polynucleotides associated with each of the different nanopore sequencing complexes;
- and
- d. characterizing each of the different nanopore variants based on one or more metrics derived from the generated sequencing data sets associated with each of the different nanopore variants, wherein the one or more metrics are based on the sequencing of the same common portion of each of the different polynucleotides associated with each of the plurality of different nanopore sequencing complexes.
2 more elements
Dependencies are inferred from the claim text.