METHOD AND SYSTEM FOR MICROBIOME-DERIVED CHARACTERIZATION, DIAGNOSTICS AND THERAPEUTICS FOR CONDITIONS ASSOCIATED WITH FUNCTIONAL FEATURES

• US 20180070825A1
• Filed: 11/16/2017
• Published: 03/15/2018
• Est. Priority Date: 10/21/2014
• Status: Active Grant

• Current Assignee: Psomagen, Inc.
• Original Assignee: uBiome, Inc.
• Inventors: Apte, Zachary, Almonacid, Daniel, Richman, Jessica
• Granted Patent: US 10,327,641 B2
• Time in Patent Office: Days
• CPC Class Codes: A61B 5/0022 Monitoring a patient using a global network, e.g. telephone networks, internet
• CPC Class Codes: A61B 5/4836 Diagnosis combined with treatment in closed-loop systems or methods A61B5/0036 takes precedence
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/112 Disease subtyping, staging or classification
• CPC Class Codes: G16B 40/00 ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
• CPC Class Codes: G16B 40/20 Supervised data analysis
• CPC Class Codes: G16B 50/00 ICT programming tools or database systems specially adapted for bioinformatics
• CPC Class Codes: G16B 50/10 Ontologies; Annotations
• CPC Class Codes: G16H 40/67 for remote operation
• CPC Class Codes: G16H 50/20 for computer-aided diagnosis, e.g. based on medical expert systems
• CPC Class Codes: G16H 50/80 for detecting, monitoring or modelling epidemics or pandemics, e.g. flu
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

A method for at least one of characterizing, diagnosing, and treating a condition associated with microbiome-derived functional features in at least a subject, the method comprising: receiving an aggregate set of biological samples from a population of subjects; generating at least one of a microbiome composition dataset and a microbiome functional diversity dataset for the population of subjects; generating a characterization of the condition based upon features extracted from at least one of the microbiome composition dataset and the microbiome functional diversity dataset; based upon the characterization, generating a therapy model configured to correct the condition; and at an output device associated with the subject, promoting a therapy to the subject based upon the characterization and the therapy model.

Claims

1. A method for at least one of characterizing, diagnosing, and treating an aminobenzoate-related condition for a subject, the method comprising:

for a set of biological samples;

determining microorganism nucleic acid sequences, comprising;

identifying primers for nucleic acid sequences associated with the aminobenzoate-related condition,

amplifying nucleic acid material from the biological samples based on the primers; and

determining alignments of the microorganism nucleic acid sequences to reference sequences associated with the aminobenzoate-related condition;

generating a microbiome functional diversity dataset based on the alignments;

extracting a set of microbiome functional diversity features from the microbiome functional diversity dataset;

generating a characterization of the aminobenzoate-related condition based on the set of microbiome functional diversity features;

determining a therapy for the subject based on the characterization and a subject biological sample from the subject; and

providing the therapy to the subject, wherein the therapy is operable to modulate microbiome composition to improve a state of the aminobenzoate-related condition.

2. The method of claim 1, further comprising:

providing a set of sampling kits to the set of subjects associated with the set of biological samples, each sampling kit of the set of sampling kits comprising a sample container operable to receive the biological sample from a collection site,

wherein determining the microorganism nucleic acid sequences comprises determining the microorganism nucleic acid sequences with a bridge amplification substrate of a next generation sequencing platform of a sample processing system, and

wherein generating the microbiome functional diversity dataset comprises generating the microbiome functional diversity dataset at computing devices operable to communicate with the next generation sequencing platform.

3. The method of claim 2, wherein the nucleic acid material comprises fragmented nucleic acid material, and wherein determining the microorganism nucleic acid sequence comprises performing, at a library preparation subsystem of the sample processing system, multiplex amplification with the fragmented nucleic acid material based on the identified primer.

4. The method of claim 1, wherein generating the characterization comprises analyzing a set of features from at least one of the set of microbiome functional diversity features and a set of microbiome composition diversity features, wherein the set of features is associated with at least one of:

relative abundance of different taxonomic groups represented in the microbiome composition diversity dataset, a ratio between at least two features associated with the different taxonomic groups and functional features, interactions between the different taxonomic groups represented in the microbiome composition diversity dataset, phylogenetic distance between the different taxonomic groups represented in the microbiome composition diversity dataset, and relative abundance between functional features from the set of microbiome functional diversity features.

5. The method of claim 1, wherein the set of microbiome functional diversity features comprises a functional feature associated with aminobenzoate degradation.

6. The method of claim 5, further comprising:

determining a modulator of a biomolecule associated with the aminobenzoate degradation; and

deriving a therapeutic composition for the condition based on the modulator,

wherein providing the therapy comprises providing the therapeutic composition to the subject based on the characterization.

7. The method of claim 1, wherein providing the therapy comprises providing a bacteriophage-based therapy to the subject, and wherein the bacteriophage-based therapy is operable to modulate a microbiome composition of the subject to improve the state of the aminobenzoate-related condition.

8. The method of claim 1, wherein generating the characterization of the aminobenzoate-related condition comprises generating the characterization associated with at least one of:

an AIDS condition, a gastric cancer condition, and a gluten intolerance condition.

9. The method of claim 8, wherein generating the characterization comprises generating the characterization based on the set of microbiome functional diversity features associated with at least one of:

a neurodegenerative diseases Kyoto Encyclopedia of Genes and Genomes (KEGG) L2 derived feature;

a transcription KEGG L2 derived feature;

a metabolism of cofactors and vitamins KEGG L2 derived feature;

an endocrine system KEGG L2 derived feature;

a cancers KEGG L2 derived feature;

an amino acid metabolism KEGG L2 derived feature;

a glycolysis/gluconeogenesis KEGG L3 derived feature;

a streptomycin biosynthesis KEGG L3 derived feature;

a restriction enzyme KEGG L3 derived feature;

a fatty acid biosynthesis KEGG L3 derived feature;

a PPAR signaling pathway KEGG L3 derived feature;

a phosphotransferase system (PTS) KEGG L3 derived feature;

a lipid metabolism KEGG L3 derived feature;

an aminobenzoate degradation KEGG L3 derived feature;

a pathways in cancer KEGG L3 derived feature;

a mismatch repair KEGG L3 derived feature;

a vitamin B6 metabolism KEGG L3 derived feature;

a butirosin and neomycin biosynthesis KEGG L3 derived feature;

a pantothenate and CoA biosynthesis KEGG L3 derived feature;

an oxidative phosphorylation KEGG L3 derived feature;

a zeatin biosynthesis KEGG L3 derived feature;

an energy metabolism KEGG L3 derived feature;

a limonene and pinene degradation KEGG L3 derived feature;

a valine, leucine, and isoleucine biosynthesis KEGG L3 derived feature;

a bacterial chemotaxis KEGG L3 derived feature;

a homologous recombination KEGG L3 derived feature;

a lipopolysaccharide biosynthesis proteins KEGG L3 derived feature;

a transcription machinery KEGG L3 derived feature;

a selenocompound KEGG L3 derived feature, a xenobiotics biodegradation and metabolism KEGG L2 derived feature;

a benzoate degradation KEGG L3 derived feature; and

an ABC.CD.P, putative ABC transport system permease protein KEGG L4 derived feature, a carbohydrate metabolism KEGG L2 derived feature;

a metabolism KEGG L2 derived feature;

a translation KEGG L2 derived feature;

a genetic information processing KEGG L2 derived feature;

an enzyme families KEGG L2 derived feature;

a replication and repair KEGG L2 derived feature;

a nucleotide metabolism KEGG L2 derived feature;

a transport and catabolism KEGG L2 derived feature;

a lipid metabolism KEGG L2 derived feature;

a signal transduction KEGG L2 derived feature;

a folding, sorting, and degradation KEGG L2 derived feature;

a cell growth and death KEGG L2 derived feature;

a biosynthesis of other secondary metabolites KEGG L2 derived feature;

an immune system diseases KEGG L2 derived feature;

an environmental adaptation KEGG L2 derived feature;

a cellular processes and signaling KEGG L2 derived feature;

a ribosome biogenesis KEGG L3 derived feature;

a peptidoglycan biosynthesis KEGG L3 derived feature;

a biosynthesis and biodegradation of secondary metabolites KEGG L3 derived feature;

a translation proteins KEGG L3 derived feature;

a pentose and glucuronate interconversions KEGG L3 derived feature;

a chromosome KEGG L3 derived feature;

a DNA repair and recombination proteins KEGG L3 derived feature;

a Glyoxylate and dicarboxylate metabolism KEGG L3 derived feature;

an inositol phosphate metabolism KEGG L3 derived feature;

a ribosome KEGG L3 derived feature;

an aminoacyl-tRNA biosynthesis KEGG L3 derived feature;

a nicotinate and nicotinamide metabolism KEGG L3 derived feature;

an amino acid related enzymes KEGG L3 derived feature;

a purine metabolism KEGG L3 derived feature;

a terpenoid backbone biosynthesis KEGG L3 derived feature;

a translation factors KEGG L3 derived feature;

a peptidases KEGG L3 derived feature;

a nucleotide excision repair KEGG L3 derived feature;

an RNA polymerase KEGG L3 derived feature;

a D-Alanine metabolism KEGG L3 derived feature;

a ribosome biogenesis in eukaryotes KEGG L3 derived feature;

a Cysteine and methionine metabolism KEGG L3 derived feature;

a Type II diabetes mellitus KEGG L3 derived feature;

a replication, recombination, and repair proteins KEGG L3 derived feature;

a pentose phosphate pathway KEGG L3 derived feature;

a pyrimidine metabolism KEGG L3 derived feature;

a carbohydrate metabolism KEGG L3 derived feature;

a fructose and mannose metabolism KEGG L3 derived feature;

a pyruvate metabolism KEGG L3 derived feature;

an other glycan degradation KEGG L3 derived feature;

a one carbon pool by folate KEGG L3 derived feature;

a DNA replication proteins KEGG L3 derived feature;

a DNA replication KEGG L3 derived feature;

a riboflavin metabolism KEGG L3 derived feature;

an other transporters KEGG L3 derived feature;

a butanoate metabolism KEGG L3 derived feature;

a sphingolipid metabolism KEGG L3 derived feature;

a galactose metabolism KEGG L3 derived feature;

a cell cycle—

Caulobacter KEGG L3 derived feature;

a Thiamine metabolism KEGG L3 derived feature;

a protein export KEGG L3 derived feature;

a glycerophospholipid metabolism KEGG L3 derived feature;

a nitrogen metabolism KEGG L3 derived feature;

a tuberculosis KEGG L3 derived feature;

a two-component system KEGG L3 derived feature;

an ethylbenzene degradation KEGG L3 derived feature;

a chloroalkane and chloroalkene degradation KEGG L3 derived feature;

a pores ion channels KEGG L3 derived feature;

a peroxisome KEGG L3 derived feature;

a sulfur metabolism KEGG L3 derived feature;

an inorganic ion transport and metabolism KEGG L3 derived feature;

an amino acid metabolism KEGG L3 derived feature;

a propanoate metabolism KEGG L3 derived feature;

an arginine and proline metabolism KEGG L3 derived feature;

a histidine metabolism KEGG L3 derived feature;

a primary immunodeficiency KEGG L3 derived feature;

a chaperones and folding catalysts KEGG L3 derived feature;

a base excision repair KEGG L3 derived feature;

an amino sugar and nucleotide sugar metabolism KEGG L3 derived feature;

a phenylpropanoid biosynthesis KEGG L3 derived feature;

a plant-pathogen interaction KEGG L3 derived feature;

a D-glutamine and D-glutamate metabolism KEGG L3 derived feature;

a polycyclic aromatic hydrocarbon degradation KEGG L3 derived feature; and

a lysine degradation KEGG L3 derived feature.

10. The method of claim 9, wherein generating the characterization comprises generating the characterization based on the set of microbiome functional diversity features and a set of microbiome composition features extracted from the microbiome composition diversity dataset, and wherein the set of microbiome composition features is associated with a set of taxa comprising at least one of:

Prevotellaceae (family), Prevotella (genus), Megasphaera (genus), Veillonellaceae (family), Erysipelotrichaceae (family), Erysipelotrichia (class), Erysipelotrichales (order), Bacteroidia (class), Bacteroidetes (phylum), Bacteroidetes/Chlorobi group (superphylum), Bacteroidales (order), Selenomonadales (order), Negativicutes (class), Lachnospiraceae (family), Flavobacteriia (class), Flavobacteriales (order), Flavobacteriaceae (family), Clostridium (genus), Coprococcus (genus), Porphyromonadaceae (family), Eubacterium ramulus (species), Oscillospiraceae (family), Acidaminococcaceae (family), Lachnospira (genus), Barnesiella (genus), Phascolarctobacterium (genus), Parasutterella (genus), Parasutterella excrementihominis (species), Bifidobacterium (genus), Moryella (genus), Dorea (genus), Oscillospira (genus), Intestinimonas (genus), Collinsella (genus), Bacteroides (genus), Dialister (genus), Subdoligranulum (genus), Hespellia (genus), Alistipes (genus), Faecalibacterium (genus), Bifidobacteriaceae (family), Bacteroidaceae (family), Ruminococcaceae (family), Coriobacteriaceae (family), Streptococcaceae (family), Rikenellaceae (family), Clostridiales Family XIII. Incertae Sedis (family), Enterobacteriaceae (family), Bifidobacteriales (order), Coriobacteriales (order), Enterobacteriales (order), Clostridiales (order), Actinobacteria (class), Clostridia (class), Actinobacteria (phylum), Firmicutes (phylum), Anaerostipes (genus), Streptococcus thermophilus (species), Clostridium leptum (species), Chlamydiae/Verrucomicrobia group (superphylum), Verrucomicrobia (phylum), Verrucomicrobiae (class), Verrucomicrobiales (order), Verrucomicrobiaceae (family), Eubacterium siraeum (species), Akkermansia (genus), Actinobacteridae (subclass), Deltaproteobacteria (class), Ruminococcus faecis (species), Desulfovibrionales (order), and delta/epsilon subdivisions (subphylum).

17. The method of claim 1, wherein the condition comprises an AIDS condition, wherein generating the characterization comprises generating the characterization based on a set of functional diversity features extracted from the microbiome functional diversity dataset, and wherein the set of functional diversity features comprises at least one of:

a neurodegenerative diseases KEGG L2 derived feature;

a transcription KEGG L2 derived feature;

a metabolism of cofactors and vitamins KEGG L2 derived feature;

an endocrine system KEGG L2 derived feature;

a cancers KEGG L2 derived feature;

an amino acid metabolism KEGG L2 derived feature;

a glycolysis/gluconeogenesis KEGG L3 derived feature;

a streptomycin biosynthesis KEGG L3 derived feature;

a restriction enzyme KEGG L3 derived feature;

a fatty acid biosynthesis KEGG L3 derived feature;

a PPAR signaling pathway KEGG L3 derived feature;

a phosphotransferase system (PTS) KEGG L3 derived feature;

a lipid metabolism KEGG L3 derived feature;

an aminobenzoate degradation KEGG L3 derived feature;

a pathways in cancer KEGG L3 derived feature;

a mismatch repair KEGG L3 derived feature;

a vitamin B6 metabolism KEGG L3 derived feature;

a butirosin and neomycin biosynthesis KEGG L3 derived feature;

a pantothenate and CoA biosynthesis KEGG L3 derived feature;

an oxidative phosphorylation KEGG L3 derived feature;

a zeatin biosynthesis KEGG L3 derived feature;

an energy metabolism KEGG L3 derived feature;

a limonene and pinene degradation KEGG L3 derived feature;

a valine, leucine, and isoleucine biosynthesis KEGG L3 derived feature;

a bacterial chemotaxis KEGG L3 derived feature;

a homologous recombination KEGG L3 derived feature;

a lipopolysaccharide biosynthesis proteins KEGG L3 derived feature;

a transcription machinery KEGG L3 derived feature; and

a selenocompound KEGG L3 derived feature.

11. A method for at least one of characterizing, diagnosing, and treating a condition for a subject, the method comprising:

receiving a biological sample from the subject;

determining a microorganism sequence based on the biological sample, comprising;

identifying a primer associated with the condition, and

amplifying nucleic acid material from the biological sample based on the identified primer;

generating a microbiome functional diversity dataset associated with aminobenzoate-related functionality, based on the microorganism sequence;

generating a characterization of the condition for the subject based on the microbiome functional diversity dataset and a characterization model associated with the condition; and

providing a therapy to the subject based on the characterization, wherein the therapy is operable to facilitate improvement of a state of the condition.

12. The method of claim 11, wherein identifying the primer comprises identifying the primer for a nucleic acid sequence associated with the condition and the benzoate-related functionality, wherein the nucleic acid material comprises fragmented nucleic acid material, and wherein amplifying the nucleic acid material comprises amplifying the fragmented nucleic acid material based on the identified primer.

13. The method of claim 11, wherein generating the characterization comprises generating the characterization based on a relative abundance feature associated with at least one of the microbiome functional diversity dataset and a microbiome composition diversity dataset, and the method further comprising generating the relative abundance feature based on at least one of:

a normalization, a feature vector derived from at least one of linear latent variable analysis and non-linear latent variable analysis, linear regression, non-linear regression, a kernel method, a feature embedding method, a machine learning method, and a statistical inference method.

14. The method of claim 11, further comprising:

determining a set of population sequences for a population of subjects based on the identified primer and a set of samples from the population of subjects;

collecting a supplementary dataset associated with diagnosis of the condition for the population of subjects;

extracting a set of microbiome functional diversity features comprising a functional feature associated with the benzoate-related functionality, based on the set of population sequences; and

generating the characterization model based on the set of microbiome functional diversity features and the supplementary dataset.

15. The method of claim 11, further comprising extracting a set of microbiome functional diversity features from the microbiome functional diversity dataset, wherein the set of microbiome functional diversity features comprises at least one of an aminobenzoate degradation KEGG L3 derived feature and a streptomycin biosynthesis KEGG L3 derived feature, and wherein generating the characterization of the condition comprises generating the characterization based on the set of microbiome functional diversity features.

16. The method of claim 11, wherein generating the characterization comprises:

extracting a set of functional diversity features from the microbiome functional diversity dataset, wherein the set of functional diversity features comprises at least one of a KEGG derived feature, a clusters of orthologous groups of proteins feature, a chemical functional feature, and a system functional feature, and wherein generating the characterization comprises generating the characterization based on the set of functional diversity features.

18. The method of claim 11, wherein the condition comprises a gastric cancer condition, wherein generating the characterization comprises generating the characterization based on a set of functional diversity features extracted from the microbiome functional diversity dataset, and wherein the set of functional diversity features comprises at least one of:

a metabolism of cofactors and vitamins KEGG L2 derived feature;

a xenobiotics biodegradation and metabolism KEGG L2 derived feature;

a benzoate degradation KEGG L3 derived feature;

an aminobenzoate degradation KEGG L3 derived feature;

a streptomycin biosynthesis KEGG L3 derived feature; and

an ABC.CD.P, putative ABC transport system permease protein KEGG L4 derived feature

19. The method of claim 11, wherein the condition comprises a gluten intolerance condition, wherein generating the characterization comprises generating the characterization based on a set of functional diversity features extracted from the microbiome functional diversity dataset, and wherein the set of functional diversity features comprises at least one of:

a carbohydrate metabolism KEGG L2 derived feature;

a metabolism KEGG L2 derived feature;

a translation KEGG L2 derived feature;

a genetic information processing KEGG L2 derived feature;

an enzyme families KEGG L2 derived feature;

a replication and repair KEGG L2 derived feature;

a nucleotide metabolism KEGG L2 derived feature;

a transport and catabolism KEGG L2 derived feature;

a lipid metabolism KEGG L2 derived feature;

a signal transduction KEGG L2 derived feature;

a neurodegenerative diseases KEGG L2 derived feature;

a metabolism of cofactors and vitamins KEGG L2 derived feature;

a xenobiotics biodegradation and metabolism KEGG L2 derived feature;

a folding, sorting, and degradation KEGG L2 derived feature;

a cell growth and death KEGG L2 derived feature;

a biosynthesis of other secondary metabolites KEGG L2 derived feature;

an immune system diseases KEGG L2 derived feature;

an environmental adaptation KEGG L2 derived feature;

a cellular processes and signaling KEGG L2 derived feature;

a ribosome biogenesis KEGG L3 derived feature;

a peptidoglycan biosynthesis KEGG L3 derived feature;

a biosynthesis and biodegradation of secondary metabolites KEGG L3 derived feature;

a translation proteins KEGG L3 derived feature;

a pentose and glucuronate interconversions KEGG L3 derived feature;

a chromosome KEGG L3 derived feature;

a DNA repair and recombination proteins KEGG L3 derived feature;

a Glyoxylate and dicarboxylate metabolism KEGG L3 derived feature;

an inositol phosphate metabolism KEGG L3 derived feature;

a ribosome KEGG L3 derived feature;

an aminoacyl-tRNA biosynthesis KEGG L3 derived feature;

a nicotinate and nicotinamide metabolism KEGG L3 derived feature;

an amino acid related enzymes KEGG L3 derived feature;

a purine metabolism KEGG L3 derived feature;

a terpenoid backbone biosynthesis KEGG L3 derived feature;

a translation factors KEGG L3 derived feature;

a peptidases KEGG L3 derived feature;

a nucleotide excision repair KEGG L3 derived feature;

an RNA polymerase KEGG L3 derived feature;

a D-Alanine metabolism KEGG L3 derived feature;

a ribosome biogenesis in eukaryotes KEGG L3 derived feature;

a Cysteine and methionine metabolism KEGG L3 derived feature;

a Type II diabetes mellitus KEGG L3 derived feature;

a homologous recombination KEGG L3 derived feature;

a replication, recombination, and repair proteins KEGG L3 derived feature;

a pentose phosphate pathway KEGG L3 derived feature;

a pyrimidine metabolism KEGG L3 derived feature;

a carbohydrate metabolism KEGG L3 derived feature;

a fructose and mannose metabolism KEGG L3 derived feature;

a pyruvate metabolism KEGG L3 derived feature;

a mismatch repair KEGG L3 derived feature;

an other glycan degradation KEGG L3 derived feature;

a one carbon pool by folate KEGG L3 derived feature;

a DNA replication proteins KEGG L3 derived feature;

a DNA replication KEGG L3 derived feature;

a riboflavin metabolism KEGG L3 derived feature;

an other transporters KEGG L3 derived feature;

a butanoate metabolism KEGG L3 derived feature;

a sphingolipid metabolism KEGG L3 derived feature;

an aminobenzoate degradation KEGG L3 derived feature;

a galactose metabolism KEGG L3 derived feature;

a cell cycle—

Caulobacter KEGG L3 derived feature;

a Thiamine metabolism KEGG L3 derived feature;

a protein export KEGG L3 derived feature;

a glycerophospholipid metabolism KEGG L3 derived feature;

a nitrogen metabolism KEGG L3 derived feature;

a tuberculosis KEGG L3 derived feature;

a two-component system KEGG L3 derived feature;

an ethylbenzene degradation KEGG L3 derived feature;

a chloroalkane and chloroalkene degradation KEGG L3 derived feature;

a pores ion channels KEGG L3 derived feature;

a peroxisome KEGG L3 derived feature;

a sulfur metabolism KEGG L3 derived feature;

an inorganic ion transport and metabolism KEGG L3 derived feature;

an amino acid metabolism KEGG L3 derived feature;

a propanoate metabolism KEGG L3 derived feature;

an arginine and proline metabolism KEGG L3 derived feature;

a histidine metabolism KEGG L3 derived feature;

a primary immunodeficiency KEGG L3 derived feature;

a chaperones and folding catalysts KEGG L3 derived feature;

a base excision repair KEGG L3 derived feature;

an amino sugar and nucleotide sugar metabolism KEGG L3 derived feature;

a phenylpropanoid biosynthesis KEGG L3 derived feature;

a plant-pathogen interaction KEGG L3 derived feature;

an energy metabolism KEGG L3 derived feature;

a streptomycin biosynthesis KEGG L3 derived feature;

a D-glutamine and D-glutamate metabolism KEGG L3 derived feature;

, a polycyclic aromatic hydrocarbon degradation KEGG L3 derived feature;

a limonene and pinene degradation KEGG L3 derived feature;

a lysine degradation KEGG L3 derived feature; and

a zeatin biosynthesis KEGG L3 derived feature.

20. The method of claim 11, wherein the condition comprises an aminobenzoate-related condition, wherein providing the therapy comprises providing an interface comprising aminobenzoate-related condition information determined based on the characterization.