Method and kit for characterizing microorganisms

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.

1 .- 20 . (canceled) 
     
     
         21 . A method of identifying a species of one or more microorganisms in a biological sample, the method comprising the steps of:
 providing a first set of primers for amplifying a first target sequence comprising hypervariable regions V1 and V2 but not V3 of a 16S rRNA gene;   providing a second set of primers for amplifying a second target sequence comprising hypervariable regions V4 and V5 but not V3 of the 16S rRNA gene;   
       a) extracting nucleic acids from the biological sample, the biological sample comprising one or more microorganisms; 
       b) amplifying the first and second target sequences from the extracted nucleic acids using the first and second set of primers; 
       c) 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; and 
       d) identifying the species of the one or more microorganisms in the biological sample based on the sequence reads of the first and second target sequences. 
     
     
         22 . The method of  claim 21 , wherein one or more primers in the first set of primers and one or more primers in the second set of primers comprises the same barcode used to identify the biological sample. 
     
     
         23 . The method of  claim 21 , wherein the biological sample is a biopsy. 
     
     
         24 . The method of  claim 21 , wherein the biological sample is a fluid, tissue or bone sample. 
     
     
         25 . The method of  claim 21 , wherein the one or more microorganisms comprise unculturable bacteria. 
     
     
         26 . The method of  claim 21 , wherein the step of sequencing comprises using an ion semiconductor sequencing platform or a platform based on stepwise addition of reversible terminator nucleotides. 
     
     
         27 . The method of  claim 21 , wherein the one or more microorganisms comprise a pathogenic community of microorganisms. 
     
     
         28 . The method of  claim 21 , wherein the predetermined length is 100 base pairs. 
     
     
         29 . The method of  claim 21 , wherein the biological sample comprises a urine sample, a blood sample, a joint sample, a bronchioalveolar lavage sample, a nasal swab sample, cerebrospinal fluid, synovial fluid, brain tissue, cardiac tissue, bone, skin, or a lymph node tissue. 
     
     
         30 . The method of  claim 21 , wherein the one or more microorganisms comprise at least one nonculturable pathogenic bacterium. 
     
     
         31 . The method  claim 21 , further comprising a step of generating a report with one or more of a genus and a species of the one or more microorganisms. 
     
     
         32 . The method of  claim 21 , wherein the predetermined criteria comprise a 95% match. 
     
     
         33 . The method of  claim 21 , wherein the second set of primers comprises at least one forward primer and at least one reverse primer. 
     
     
         34 . The method of  claim 21 , wherein the species of the one or more microorganisms is identified by
 (i) comparing each of the sequence reads having greater than or equal to a predetermined length to data in a library; and   (ii) 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.   
     
     
         35 . The method of  claim 34 , wherein the determining of step (ii) comprises for each sequence read of the sequence reads determined to match a reference sequence in the library whether a confidence of the match is within a predetermined confidence percentage. 
     
     
         36 . The method of  claim 35 , wherein the confidence percentage is 95%.