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Publication Number
US-9914979-B2
Publication Date
2018-03-13
Expiration Date
Abstract
The present disclosure provides methods of characterizing one or more microorganisms and kits for characterizing at least one microorganism. Exemplary methods include preparing an amplicon library, sequencing a characteristic gene sequence to obtain a gene sequence, and characterizing the one or more microorganisms based on the gene sequence using a computer-based genomic analysis of the gene sequence. Exemplary kits include at least one forward primer including an adapter sequence and a priming sequence, for a target sequence, and at least one reverse primer.
Core Innovation
Species identification is achieved by selectively amplifying two 16S rRNA gene target sequences from extracted nucleic acids in a human clinical sample. The first target sequence comprises hypervariable region V1 and hypervariable region V2 and does not include hypervariable region V3, and the second target sequence comprises hypervariable region V5 and hypervariable region V4 and does not include hypervariable region V3.
The target sequences are sequenced to obtain sequence reads from 5′ to 3′ V1 and V2 of the first target sequence and from 5′ to 3′ V5 and V4 of the second target sequence. Species are identified by comparing each sequence read having greater than or equal to a predetermined length to data in a library and determining sequence reads that correspond to data in the library based on predetermined criteria.
Claims Coverage
The consolidated content provides two independent claims covering a species-identification method for one or more microorganisms in a human clinical sample using two 16S rRNA gene target sequences that exclude hypervariable region V3, followed by sequencing and library-based read comparison using predetermined criteria. The claims present the same overall inventive framework, with different specified primer sets.
Two 16S rRNA target sequences excluding V3 for selective amplification
Preparing from a 16S rRNA gene first and second target sequences by contacting extracted nucleic acids with a first set of primers for selective amplification of a first target sequence comprising hypervariable region V1 and hypervariable region V2, wherein the first target sequence does not include hypervariable region V3, and contacting with a second set of primers for selective amplification of a second target sequence comprising hypervariable region V5 and hypervariable region V4, wherein the second target sequence does not include hypervariable region V3, and amplifying the first and second target sequences.
Sequencing V1+V2 and V5+V4 to obtain sequence reads
Sequencing from 5′ to 3′ V1 and V2 of the first target sequence and sequencing from 5′ to 3′ V5 and V4 of the second target sequence to obtain sequence reads of the first and second target sequences.
Library comparison using predetermined criteria to identify species
Identifying the species of the one or more microorganisms based on the sequence reads by comparing each sequence read having greater than or equal to a predetermined length to data in a library, and determining sequence reads that correspond to data in the library based on predetermined criteria, wherein the sequence reads identify the species of the one or more microorganisms.
Primer set specification for the first and second target sequences
Preparing the first and second target sequences using specified primer sets, including SEQ ID NOs:33 and 35-50 in one claim and SEQ ID NOs:34 and 51-66 in another claim, with both target sequences excluding hypervariable region V3.
Species identification is carried out by selectively amplifying two 16S rRNA gene target sequences that exclude V3, sequencing those regions, and identifying species through library comparison using predetermined criteria. The independent claims differ in the specific primer set details recited for target amplification.
Stated Advantages
Documented Applications
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