Automated priming and library loading device

Abstract

Provided herein are automated apparatus and methods 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 library preparation and sequencing.

What is claimed is: 
     
         1 . A library preparation system comprising:
 (a) a platform comprising a plurality of containers, wherein each container of a first subset of said plurality of containers comprises a sample, wherein said sample comprises an isolated nucleic acid sequence, and each container of a second subset of said plurality of containers comprises a plurality of reagents;   (b) a fluid handling system comprising a robotic arm configured to withdraw and dispense (i) said sample from said first subset of said plurality of containers or (ii) said plurality of reagents from said second subset of said plurality of containers;   (c) an automated translation stage disposed above said platform and coupled to said fluid handling system; and   (d) a controller communicatively coupled to said fluid handling system and said automated translation stage.   
     
     
         2 . The library preparation system of  claim 1 , further comprising: a library preparation chamber comprising a heating element and a thermometer, and wherein said heating element is configured to cycle a temperature of said platform between a first temperature and a second temperature. 
     
     
         3 . The library preparation system of  claim 1 , further comprising a sequencing flow cell that is configured for a pore sequencing reaction. 
     
     
         4 . The library preparation system of  claim 1 , further comprising a sequencing flow cell that is configured for a sequencing-by-synthesis reaction. 
     
     
         5 . The library preparation system of  claim 1 , further comprising a sequencing chamber comprising a sequencing flow cell, wherein said robotic arm is configured to move said mixture to said sequencing chamber. 
     
     
         6 . The library preparation system of  claim 5 , wherein said sequencing chamber is disposed on said platform. 
     
     
         7 . The library preparation system of  claim 1 , wherein said fluid handling system further comprises one or more pumps or compressors. 
     
     
         8 . The library preparation system of  claim 2 , wherein the controller comprises instructions configured to implement a method, wherein said instructions comprise cycling, by said heating element, said temperature of said platform between said first temperature and said second temperature in an amplification reaction of at least a portion of said isolated nucleic acid sequence. 
     
     
         9 . The library preparation system of  claim 1 , wherein the controller comprises instructions, wherein said instructions further comprise barcoding said isolated nucleic acid sequence by coupling said one or more barcode nucleic acid sequences to at least a portion of said isolated nucleic acid sequence. 
     
     
         10 . The library preparation system of  claim 1 , further comprising a mechanical mixer comprising a motor mechanically coupled to said plurality of containers, wherein said mechanical mixer is configured to agitate said plurality of containers. 
     
     
         11 . The library preparation system of  claim 1 , wherein the controller comprises instructions configured to implement a method, wherein said instructions comprise activating said mechanical mixer to agitate said plurality of containers, thereby mixing said sample with said plurality of reagents to produce said mixture. 
     
     
         12 . The library preparation system of  claim 1 , wherein said plurality of reagents further comprises a nucleic acid primer. 
     
     
         13 . The library preparation system of  claim 2 , wherein said instructions further comprise:
 sensing a third temperature proximal to said platform that is below a set temperature;   turning on said heating element;   sensing a fourth temperature proximal to said platform that is said set temperature; and   turning off said heating element.   
     
     
         14 . The library preparation system of  claim 1 , further comprising an antifouling coating coupled to an interior surface of said fluid handling system. 
     
     
         15 . The library preparation system of  claim 1 , further comprising a gas cylinder comprising a gas, wherein said gas cylinder is fluidically connected with said fluid handling system, and wherein said fluid handling system is configured to spray said first subset of said plurality of containers with said gas. 
     
     
         16 . The library preparation system of  claim 1 , wherein said second subset of said plurality of containers comprises (i) a first container comprising said a first barcode nucleic acid sequence of said one or more barcode nucleic acid sequences, and (ii) a second container comprising a second barcode nucleic acid sequence of said one or more barcode nucleic acid sequences, wherein said first barcode nucleic acid sequence is different than said second barcode nucleic acid sequence. 
     
     
         17 . The library preparation system of  claim 16 , wherein said one or more barcode nucleic acid sequences in each container of said second subset of said plurality of containers is different than said one or more barcode nucleic acid sequences in another container of said second subset of said plurality of containers. 
     
     
         18 . The library preparation system of  claim 16 , wherein each container of said second subset of said plurality of containers comprises two or more types of said one or more barcode nucleic acid sequences. 
     
     
         19 . The library preparation system of  claim 16 , wherein said one or more or more barcode nucleic acid sequences comprises at least three distinct barcode nucleic acid sequences comprising a first barcode nucleic acid sequence, a second barcode nucleic acid sequence, and a third barcode nucleic acid sequence, and wherein said combining said sample with said plurality of reagents to produce said mixture comprises:
 adding said first barcode nucleic acid sequence to a first plurality of said isolated nucleic acid sequence in said sample, thereby providing a first plurality of indexed nucleic acid sequences;   adding a second barcode nucleic acid sequence to said first plurality of said isolated nucleic acid sequence in said sample, thereby providing a second plurality of indexed nucleic acid sequences; and   adding a third barcode nucleic acid sequence to said first plurality of said isolated nucleic acid sequence in said sample, thereby providing a third plurality of indexed nucleic acid sequences.   
     
     
         20 . The library preparation system of  claim 19 , wherein said instructions further comprise:
 performing, by said sequencing chamber, a sequencing reaction on said third plurality of said isolated nucleic acid sequence; and   demultiplexing, by said controller, said third plurality of said isolated nucleic acid sequence comprising said first barcode nucleic acid sequence, said second barcode nucleic acid sequence, and said third barcode nucleic acid sequence.   
     
     
         21 . A method of preparing a library comprising:
 inserting a first sample comprising a nucleic acid into a library preparation chamber, wherein the first sample is comprised within a container;   mixing the first sample with a first reagent using a fluid handling system;   withdrawing the first sample from the container using the fluid handling system; and   inserting the first sample into a flow cell.   
     
     
         22 . The method of  claim 21 , wherein the inserting is performed under conditions sufficient to prevent failure of the flow cell due to introduction of air into the flow cell. 
     
     
         23 . The method of  claim 21 , wherein the conditions sufficient to prevent failure of the flow cell due to introduction of air into the flow cell comprise not introducing air bubbles into a flow path into the flow cell. 
     
     
         24 . The method of  claim 21 , further comprising introducing, by the fluid handling system, a conditioning liquid into the flow cell to remove obstructions in the flow path of the flow cell. 
     
     
         25 . The method of  claim 24 , further comprising introducing, by the fluid handling system, a buffer solution into the flow cell to displace the conditioning liquid in the flow path of the flow cell. 
     
     
         26 . The method of  claim 25 , further comprising creating a continuous fluid channel in the flow path of the flow cell sufficient to prevent the failure of the flow cell due to the introduction of air into the flow path of the flow cell. 
     
     
         27 . The method of  claim 26 , further comprising flushing the flow cell with a fluid dispensed from the fluid handling system following inserting the sample into the flow cell. 
     
     
         28 . The method of  claim 23 , wherein inserting the first sample into a flow cell comprises dispensing, by the fluid handling system, a 3 microliter to 50 microliter volume of the sample into a sample input port of the flow cell. 
     
     
         29 . The method of  claim 28 , further comprising contacting the sample input port with the fluid handling system prior to inserting the sample into the sample input port of the flow cell. 
     
     
         30 . The method of  claim 21 , further comprising adding at least one barcoded nucleic acid sequence to the sample and mixing the sample prior to inserting the first sample into the flow cell.