Methods for species-level resolution of microorganisms
Inventors
KEMP, Ryan • Weinstein, Mike • Tang, Shuiquan
Assignees
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Publication Number
US-12473601-B2
Publication Date
2025-11-18
Expiration Date
Abstract
Methods and devices are provided for the identification and/or quantification of microbes. In particular, the methods comprise: (a) extracting DNA from a microbial sample; (b) performing 16S ribosomal RNA gene-targeted sequencing to obtain DNA sequences; and (c) analyzing the DNA sequences to identify the species of the one or more microbes.
Core Innovation
The invention relates to a method for the identification and/or quantification of one or more microbes from a microbial sample. The method comprises extracting DNA from the microbial sample and performing 16S ribosomal RNA gene-targeted sequencing to obtain DNA sequences, including amplifying the V1-V3 region and/or the V3-V4 region of the 16S ribosomal RNA gene.
The obtained DNA sequences are analyzed to identify the species of the one or more microbes. The analysis includes trimming and filtering using FIGARO and denoising/inference using DADA2 to obtain amplicon sequences, and taxonomy assignment is performed using species-resolving approaches that avoid clustering into operational taxonomic units (OTUs).
The method supports species-level identification and/or quantification across diverse sample types, including human and environmental samples. The document further describes improving taxonomic accuracy by using curated references and handling unnamed species using “species IDs”.
Claims Coverage
The independent claim is clm-00001. The claim set includes 8 inventive features, with additional limitations on specific amplicon regions, FIGARO and DADA2 analysis steps, exclusion of culture/array analysis, taxonomy assignment without OTU clustering, and a processing-time threshold.
DNA extraction from a microbial sample
Extracting DNA from a microbial sample.
16S ribosomal RNA gene-targeted sequencing of V1-V3 and/or V3-V4
Performing 16S ribosomal RNA gene-targeted sequencing with amplifying the V1-V3 region and/or means for amplifying the V3-V4 region, thereby obtaining DNA sequences.
Species identification of one or more microbes from analyzed DNA sequences
Analyzing the DNA sequences to identify the species of the one or more microbes.
Excluding microbial culture and array analysis
The method does not comprise microbial culture or array analysis.
FIGARO trimming of amplicon sequences
The bioinformatics analysis comprises using FIGARO to trim amplicon sequences.
FIGARO trimming followed by DADA2 inference of amplicon sequences
The bioinformatics analysis uses FIGARO to trim amplicon sequences and then DADA2 to infer amplicon sequences.
Taxonomy assignment without OTU clustering
Taxonomy assignment does not comprise clustering amplicon sequences into operational taxonomic units (OTUs).
Processing in less than 3 days
The method is performed in less than 3 days.
Overall, the claim coverage centers on species-level identification and/or quantification by combining DNA extraction, 16S ribosomal RNA gene-targeted sequencing of V1-V3 and/or V3-V4 regions, and downstream FIGARO trimming with DADA2 inference. Taxonomy assignment is constrained to avoid OTU clustering, and the method includes explicit workflow exclusions and a processing-time threshold.
Stated Advantages
Improved species-level resolution compared with a QIIME/Greengenes pipeline on mock/community and fecal datasets.
Ability to resolve previously unknown Lachnospiraceae species.
Documented Applications
Human and environmental sample types for microbial identification and/or quantification.
Testing on mock/community and fecal datasets.
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