Automated priming and library loading device

Abstract

Provided herein are automated apparatus for the identification of microorganisms in various samples. The disclosure solves existing challenges encountered in identifying and distinguishing various types of microorganisms, including viruses and bacteria in a timely, efficient, and automated manner by sequencing.

What is claimed is: 
     
         1 . A method of analyzing a biological sample, the method comprising:
 (a) performing pore sequencing of a plurality of nucleic acid sequences from a biological sample; and   (b) performing an assay on the biological sample if the pore sequencing identifies a threshold level of nucleic acid sequences from a microorganism in the biological sample.   
     
     
         2 . The method of  claim 1 , wherein the performing the pore sequencing in (a) is performed for less than 30 minutes. 
     
     
         3 . The method of  claim 1 , wherein the performing the pore sequencing in (a) is performed for less than 20 minutes. 
     
     
         4 . The method of  claim 1 , wherein the threshold is no more than 0.1% of the nucleic acid sequences from the microorganism. 
     
     
         5 . The method of  claim 1 , further comprising performing an amplification reaction on the plurality of nucleic acid sequences prior to the performing of the pore sequencing in (a). 
     
     
         6 . The method of  claim 1 , wherein the assay comprises a serotyping assay, a culturing assay, a Pulse Field Gel Electrophoresis (PFGE) assay, a Bioprinter® assay, a q-PCR assay, a Sanger sequencing assay, an ELISA assay, a Whole Genome Sequencing (WGS) assay, a targeted sequencing assay, or a shotgun metagenomics assay, or any combination thereof. 
     
     
         7 . The method of  claim 1 , wherein the microorganism comprises a microorganism of the  Salmonella  genus, a microorganism of the  Campylobacter  genus, a microorganism of the  Listeria  genus, or a microorganism of the  Escherichia  genus, or any combination thereof. 
     
     
         8 . The method of  claim 7 , wherein the microorganism of the  Salmonella  genus has a serotype comprising Enteritidis, Typhimurium, Newport, Javiana, Infantis, Montevideo, Heidelberg, Muenchen, Saintpaul, Oranienburg, Braenderup, Paratyphi B var. L(+) Tartrate+, Agona, Thompson, or Kentucky. 
     
     
         9 . The method of  claim 7 , wherein the microorganism of the  Escherichia  genus has a serotype comprising O103, O111, O121, O145, O26, O45, or O157. 
     
     
         10 . The method of  claim 7 , wherein the microorganism of the  Listeria  genus has a serotype comprising 2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4ab, 4c, 4d, or 4e. 
     
     
         11 . The method of  claim 7 , wherein the microorganism of the  Campylobacter  genus comprises  C. jejunis, C. lari , or  C. coli.    
     
     
         12 . The method of  claim 7 , further comprising distinguishing between a normal level of the microorganism of the  Campylobacter  genus and a hazardous level of microorganism of the  Campylobacter  genus in the biological sample. 
     
     
         13 . The method of  claim 7 , further comprising distinguishing between a first strain of the microorganism of the  Campylobacter  genus and a second strain of the microorganism of the  Campylobacter  genus. 
     
     
         14 . The method of  claim 1 , wherein the biological sample comprises a food sample. 
     
     
         15 . The method of  claim 1 , wherein the biological sample comprises a surface swab of an environment associated with a food sample. 
     
     
         16 . The method of  claim 1 , wherein the biological sample is a non-food sample. 
     
     
         17 . The method of  claim 1 , wherein the biological sample comprises a swab of a surface rinse of an environment associated with the biological sample. 
     
     
         18 . The method of  claim 1 , wherein the performing the pore sequencing in (a) is performed on the threshold level with greater than 90% sensitivity. 
     
     
         19 . The method of  claim 18 , wherein the threshold level comprises between 1% and 25% of the nucleic acids from the microorganism. 
     
     
         20 . The method of  claim 1 , wherein the performing the pore sequencing in (a) is performed on the threshold level of the nucleic acid sequences of the microorganism with greater than 90% specificity. 
     
     
         21 . The method of  claim 20 , wherein the threshold level comprises between 1% and 25% of the nucleic acids from the microorganism. 
     
     
         22 . The method of  claim 1 , wherein the assay on the biological sample in (b) determines if the microorganism is alive or dead. 
     
     
         23 . The method of  claim 22 , wherein the performing the assay on the biological sample comprises:
 (i) adding a photoreactive DNA-binding dye or a DNA intercalating reagent to the sample; and   (ii) culturing the sample.   
     
     
         24 . The method of  claim 22 , wherein the performing the assay on the biological sample comprises adding a reagent to the sample which interacts with nucleic acids in a dead microorganism and does not interact with nucleic acids in a live microorganism. 
     
     
         25 . The method of  claim 22 , wherein the performing the assay on the biological sample comprises analyzing a methylation or other epigenetic pattern of the nucleic acids from the microorganism in the biological sample. 
     
     
         26 . The method of  claim 1 , wherein the performing the pore sequencing in (a) is performed with a flow cell, and wherein the performing the pore sequencing in (a) comprises reusing the flow cell by performing another pore sequencing reaction on a second plurality of nucleic acid sequences from a second biological sample using the flow cell. 
     
     
         27 . The method of  claim 1 , wherein the performing the pore sequencing in (a) comprises:
 (i) adding a first molecular index to a first subset of the plurality of nucleic acid sequences, thereby providing a first plurality of indexed nucleic acid sequences;   (ii) adding a second molecular index to the first subset of the plurality of nucleic acid sequences, thereby providing a second plurality of indexed nucleic acid sequences;   (iii) adding a third molecular index to the first subset of the plurality of nucleic acid sequences from the sample, thereby providing a third plurality of indexed nucleic acid sequences;   (iv) performing a sequencing reaction on the third plurality of nucleic acid sequences; and   (v) demultiplexing, by a computer system, the third plurality of nucleic acid sequences comprising the first molecular index, the second molecular index, and the third molecular index.   
     
     
         28 . The method of  claim 27 , wherein the first molecular index, the second molecular index, or the third molecular index comprises a sequence of nucleotides has a design comprising a nonperiodic design. 
     
     
         29 . The method of  claim 27 , wherein the performing the pore sequencing in (a) is performed with a flow cell, and wherein the performing the pore sequencing in (a) further comprises reusing the flow cell by performing another pore sequencing reaction on a second plurality of nucleic acid sequences from a second biological sample using the flow cell. 
     
     
         30 . The method of  claim 29 , wherein the demultiplexing reduces an amount of crosstalk between the first molecular index, the second molecular index, and the third molecular index when the flow cell is reused.