SYSTEMS AND METHODS FOR CONTINUOUS FLOW PCR SYSTEMS

A liquid handling system of a PCR system is instructed to obtain a matrix of samples and reagents for a PCR experiment. A fluid pumping system of the PCR system is instructed to maintain a continuous flow of a transport fluid through a plurality of micro-channels that allows the mixture of the samples and the reagents producing a plurality of mixed sample droplets. One or more post-bridge detection values are received from a post-bridge detection system of the PCR system for each mixed sample droplet to determine if the mixed sample droplet is mixed correctly. A thermocycler of the PCR system is instructed to maintain one or more temperatures for cycling the temperature of the plurality of mixed sample droplets. One or more endpoint detection values are received from an endpoint detection system of the PCR system for each mixed sample droplet to analyze the PCR experiment.

INTRODUCTION

Polymerase chain reaction (PCR) systems or thermocyclers typically include a sample block, a heated cover, and heating and cooling elements. These components are then controlled or monitored by an onboard control system. Real-time PCR systems or thermocyclers generally also include an optical detection system for detecting electromagnetic radiation emitted by one or more probes attached to a nucleic acid sample. Real-time PCR systems can additionally include an external computer or control system for controlling and monitoring system components and analyzing data produced by the optical detection system.

Current standard PCR systems and real-time PCR systems are well-based systems. These systems receive samples in a sample support device that includes a plurality of wells. The samples are prepared or mixed with reagents before being loaded into the PCR system. The PCR system then cycles the temperatures of the samples in the wells. Additionally, real-time PCR systems monitor the samples in the wells for electromagnetic or fluorescent emissions.

As the uses and need for genetic and genomic information have increased, so has the need for PCR amplification and analysis. In particular, it has become increasingly important to improve the throughput of PCR systems. Although each generation of PCR systems can cycle the temperatures of samples slightly faster, the technology has not kept up with the performance improvements of other genetic and genomic analysis instruments. For example, deoxyribonucleic acid (DNA) sequencing instruments are advancing to the point where sample preparation and PCR amplification are the most limiting steps in terms of time and cost for sequencing experiments.

In addition, the reliance of current PCR systems on well-based technology limits the overall throughput of these systems. Current systems can cycle the temperatures of samples in approximately 40 minutes. Using the largest well-based sample support device with 384 wells, therefore, produces a maximum overall sample throughput of about 500 samples per hour. Further, current PCR systems receive samples already prepared or mixed in the sample support device. Therefore these systems are dependent on the time consuming and sometimes manual step of well-based sample preparation.

SUMMARY

A system, method, and computer program product are provided for high throughput polymerase chain reaction (PCR) amplification and analysis. The system includes a PCR system and a processor in communication with the PCR system. The method includes steps that use a PCR system and a processor.

The computer program product includes a non-transitory and tangible computer-readable storage medium. The computer-readable storage medium includes a program with instructions that are executed on a processor. The instructions executed on the processor perform a method for high throughput PCR amplification and analysis. The method includes providing a system of distinct software modules that includes a liquid handling module, a fluid pumping module, a post-bridge detection module, a thermocycler module, and an endpoint detection module.

In the system and method, a processor sends instructions to and receives data values from a number of components of the PCR system. The processor instructs a liquid handling system to obtain a plurality of samples and a plurality of reagents for a PCR experiment. The processor instructs a fluid pumping system to maintain a continuous flow of a transport fluid through a plurality of micro-channels. The continuous flow allows the fluid pumping system to receive the plurality of samples and the plurality of reagents from the liquid handling system as droplets in the plurality of micro-channels. The continuous flow also allows the fluid pumping system to mix the plurality of samples and the plurality of reagents using the geometry of the plurality of micro-channels producing a plurality of mixed sample droplets in the plurality of micro-channels.

The processor receives from a post-bridge detection system of the PCR system one or more post-bridge detection values for each mixed sample droplet of the plurality of mixed sample droplets to determine if each mixed sample droplet is mixed correctly. The processor instructs a thermocycler of the PCR system to maintain one or more temperatures for cycling the temperature of the plurality of mixed sample droplets in the plurality of micro-channels. Finally, the processor receives from an endpoint detection system of the PCR system one or more endpoint detection values for each mixed sample droplet of the plurality of mixed sample droplets to analyze the PCR experiment.

In various embodiments, the processor instructs the liquid handling system to pipette samples from a first sample support device located on a first tray of the liquid handling system, pipette assay reagents from a second sample support device located on a second tray of the liquid handling system, and pipette a master mix reagent from a vessel.

In various embodiments, the one or more post-bridge detection values include a time stamp of the mixed sample droplet. In various embodiments, the one or more post-bridge detection values include the intensity of electromagnetic radiation absorbed or reflected by the mixed sample droplet. In various embodiments, the one or more post-bridge detection values include a first intensity of electromagnetic radiation emitted by a first dye of a sample of the mixed sample droplet, a second intensity of electromagnetic radiation emitted by a second dye of an assay reagent of the mixed sample droplet, and a third intensity of electromagnetic radiation emitted by a third dye of a master mix reagent of the mixed sample droplet.

In various embodiments, the processor further instructs the liquid handling system to re-sample a sample and an assay reagent of the mixed sample droplet, if the processor determines from the one or more post-bridge detection values that the mixed sample droplet is mixed incorrectly.

In various embodiments, the one or more endpoint detection values include a location of a micro-channel and a spectral intensity detected from the micro-channel.

These and other features of the present teachings are set forth herein.

Before one or more embodiments of the present teachings are described in detail, one skilled in the art will appreciate that the present teachings are not limited in their application to the details of construction, the arrangements of components, and the arrangement of steps set forth in the following detailed description or illustrated in the drawings. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

DESCRIPTION OF VARIOUS EMBODIMENTS

FIG. 1is a block diagram that illustrates a computer system100, upon which embodiments of the present teachings may be implemented. Computer system100includes a bus102or other communication mechanism for communicating information, and a processor104coupled with bus102for processing information. Computer system100also includes a memory106, which can be a random access memory (RAM) or other dynamic storage device, coupled to bus102for determining base calls, and instructions to be executed by processor104. Memory106also may be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor104. Computer system100further includes a read only memory (ROM)108or other static storage device coupled to bus102for storing static information and instructions for processor104. A storage device110, such as a magnetic disk or optical disk, is provided and coupled to bus102for storing information and instructions.

Computer system100may be coupled via bus102to a display112, such as a cathode ray tube (CRT) or liquid crystal display (LCD), for displaying information to a computer user. An input device114, including alphanumeric and other keys, is coupled to bus102for communicating information and command selections to processor104. Another type of user input device is cursor control116, such as a mouse, a trackball or cursor direction keys for communicating direction information and command selections to processor104and for controlling cursor movement on display112. This input device typically has two degrees of freedom in two axes, a first axis (i.e., x) and a second axis (i.e., y), that allows the device to specify positions in a plane.

A computer system100can perform the present teachings. Consistent with certain implementations of the present teachings, results are provided by computer system100in response to processor104executing one or more sequences of one or more instructions contained in memory106. Such instructions may be read into memory106from another computer-readable medium, such as storage device110. Execution of the sequences of instructions contained in memory106causes processor104to perform the process described herein. Alternatively hard-wired circuitry may be used in place of or in combination with software instructions to implement the present teachings. Thus implementations of the present teachings are not limited to any specific combination of hardware circuitry and software.

The term “computer-readable medium” as used herein refers to any media that participates in providing instructions to processor104for execution. Such a medium may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Non-volatile media includes, for example, optical or magnetic disks, such as storage device110. Volatile media includes dynamic memory, such as memory106. Transmission media includes coaxial cables, copper wire, and fiber optics, including the wires that comprise bus102.

Common forms of computer-readable media include, for example, a floppy disk, a flexible disk, hard disk, magnetic tape, or any other magnetic medium, a CD-ROM, any other optical medium, punch cards, papertape, any other physical medium with patterns of holes, a RAM, PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.

Various forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to processor104for execution. For example, the instructions may initially be carried on the magnetic disk of a remote computer. The remote computer can load the instructions into its dynamic memory and send the instructions over a telephone line using a modem. A modem local to computer system100can receive the data on the telephone line and use an infra-red transmitter to convert the data to an infra-red signal. An infra-red detector coupled to bus102can receive the data carried in the infra-red signal and place the data on bus102. Bus102carries the data to memory106, from which processor104retrieves and executes the instructions. The instructions received by memory106may optionally be stored on storage device110either before or after execution by processor104.

The following descriptions of various implementations of the present teachings have been presented for purposes of illustration and description. It is not exhaustive and does not limit the present teachings to the precise form disclosed. Modifications and variations are possible in light of the above teachings or may be acquired from practicing of the present teachings. Additionally, the described implementation includes software but the present teachings may be implemented as a combination of hardware and software or in hardware alone. The present teachings may be implemented with both object-oriented and non-object-oriented programming systems.

Systems and Methods of Data Processing

Continuous Flow PCR System

As described above, the reliance of current polymerase chain reaction (PCR) systems on well-based technology can limit the overall throughput of these systems. Also, current PCR systems receive samples already prepared or mixed in the sample support device. Therefore these systems are dependent on the time consuming and sometimes manual step of well-based sample preparation.

In various embodiments, systems and methods for continuous flow PCR amplification and analysis are used. These systems and methods significantly increase the sample throughput of a PCR experiment and reduce the limitations imposed by well-based technology. In particular, systems and methods for continuous flow PCR essentially eliminate a sample preparation step by incorporating it into the PCR process.

FIG. 2is a schematic diagram showing a system200for high throughput PCR amplification and analysis, in accordance with various embodiments. System200includes PCR system210and processor220. PCR system210, in turn, includes liquid handling system230, fluid pumping system240, post-bridge detection system250, thermocycler260, and endpoint detection system270.

Processor220is in communication with PCR system210. Processor220can include, but is not limited to, a computer, a microprocessor, a microcontroller, an application specific integrated circuit (ASIC), or any device capable of executing instructions and sending and receiving data or control communications.

Processor220instructs liquid handling system230to obtain a plurality of samples and a plurality of reagents for a PCR experiment. In various embodiments, processor220instructs liquid handling system230to pipette samples from a first sample support device (not shown) located on tray231of liquid handling system230, pipette assay reagents from a second sample support device (not shown) located on tray232of liquid handling system230, and pipette a master mix reagent from vessel233.

In various embodiments, a sample support device may be a glass or plastic slide with a plurality of sample regions. Some examples of a sample support device may include, but are not limited to, a multi-well plate, such as a standard microtiter 96-well, a 384-well plate, or a microcard, or a substantially planar support, such as a glass or plastic slide. The sample regions in various embodiments of a sample support device may include depressions, indentations, ridges, and combinations thereof, patterned in regular or irregular arrays formed on the surface of the substrate.

Processor220instructs fluid pumping system240to maintain a continuous flow of a transport fluid through a plurality of micro-channels. The transport fluid or oil is a passive buffer for carrying samples around system200.FIG. 2shows a single micro-channel of the plurality of micro-channels. This single micro-channel or tube includes draft line241and thermocycler line242. Draft line241is used to bleed off excess transport fluid and maintain the continuous flow of a transport fluid through the micro-channel at a constant flow rate. Thermocycler line242is used to carry mixed samples through system200.

Processor220instructs fluid pumping system240to maintain a continuous flow of a transport fluid in order to receive the plurality of samples and the plurality of reagents from liquid handling system230as droplets in the plurality of micro-channels. The continuous flow of a transport fluid by fluid pumping system240draws a sample droplet from tip235of liquid handling system230up through line245of fluid pumping system240. Similarly, the continuous flow of a transport fluid by fluid pumping system240draws an assay reagent droplet from tip236of liquid handling system230up through line246of fluid pumping system240and draws a master mix reagent droplet from tip237of liquid handling system230up through line247of fluid pumping system240, for example.

Further, the continuous flow of a transport fluid by fluid pumping system240causes the plurality of samples and the plurality of reagents to be mixed using the geometry of the plurality of micro-channels. This results in a plurality of mixed sample droplets in the plurality of micro-channels. The geometry of the plurality of micro-channels that causes the plurality of samples and the plurality of reagents to be mixed is a junction or liquid bridge of micro-channels, for example.

Junction249is an exemplary liquid bridge for mixing samples and reagents for a single micro-channel. Lines245,246, and247meet at junction249. Through precise timing control, processor220instructs liquid handling system230to select sample, assay reagent, and master mix droplets using tips235,236, and247at specific times so that fluid pumping system240draws these droplets to junction249at the same time. Because sample, assay reagent, and master mix droplets reach junction249simultaneously, they are mixed as they are moving with the continuous flow of transport fluid. The mixture produces a mixed sample droplet. This mixed sample droplet leaves junction249and enters thermocycler line242. The mixed sample droplet continues moving with the continuous flow of transport fluid at a constant flow rate in thermocycler line242.

In order to determine if each mixed sample droplet is mixed correctly, processor220receives one or more post-bridge detection values for each mixed sample droplet of the plurality of mixed sample droplets from post-bridge detection system250. Post-bridge detection system250, for example, detects mixed sample droplets in thermocycler line242at precise time steps selected by processor220. In various embodiments, post-bridge detection system250is an optical system that includes one or more sources of illumination and one or more cameras. In various embodiments, one camera is used and the one or more post-bridge detection values include the intensity of electromagnetic radiation absorbed or reflected by each mixed sample droplet.

In various embodiments, three cameras are used by post-bridge detection system250. The one or more post-bridge detection values received by processor220then include a first intensity of electromagnetic radiation emitted by a first dye of a sample of each mixed sample droplet, a second intensity of electromagnetic radiation emitted by a second dye of an assay reagent of each mixed sample droplet, and a third intensity of electromagnetic radiation emitted by a third dye of a master mix reagent of the mixed sample droplet. In various embodiments, the one or more post-bridge detection values also include a time stamp of the mixed sample droplet so the processor can identify the sample and reagents used to create the mixed sample droplet.

In various embodiments, processor220instructs liquid handling system230to re-sample a sample and an assay reagent of a mixed sample droplet, if processor220determines from the one or more post-bridge detection values that the mixed sample droplet is mixed incorrectly. In other words, if processor220determines that the one or more post-bridge detection values that the mixed sample droplet are not indicative of a proper mixture, processor instructs liquid handling system230to re-sample the sample and reagents used to create the mixed sample droplet.

After a mixed sample droplet of the plurality of mixed sample droplets is analyzed by post-bridge detection system250, it moves to thermocycler260. Processor220instructs thermocycler260to maintain one or more temperatures for cycling the temperature of the plurality of mixed sample droplets in the plurality of micro-channels. In various embodiments, thermocycler260includes two or more heating and cooling elements that are instructed to maintain two or more temperatures. As each mixed sample droplet is moved among the two or more heating and cooling elements, the temperature of the mixed sample droplet is cycled.

Finally, processor220receives from endpoint detection system270one or more endpoint detection values for each mixed sample droplet of the plurality of mixed sample droplets. Processor220uses the one or more endpoint detection values to analyze the PCR experiment. In various embodiments, endpoint detection system270is also an optical detection system. Endpoint detection system270is a hyperspectral imaging system that determines both spatial and spectral information, for example. Therefore, in various embodiments, the one or more endpoint detection values include the location of a micro-channel and a spectral intensity value detected from that micro-channel. The location of the micro-channel allows processor220to identify the mixed sample droplet and the spectral intensity value detected provides a measure of the result of the PCR experiment.

FIG. 3is an exemplary flowchart showing a method300for high throughput PCR amplification and analysis, in accordance with various embodiments.

In step310of method300, a liquid handling system of a PCR system is instructed to obtain a plurality of samples and a plurality of reagents for a PCR experiment using a processor.

In step320, a fluid pumping system of the PCR system is instructed to maintain a continuous flow of a transport fluid through a plurality of micro-channels using the processor. The continuous flow allows the fluid pumping system to receive the plurality of samples and the plurality of reagents from the liquid handling system as droplets in the plurality of micro-channels. The continuous flow also allows the fluid pumping system to mix the plurality of samples and the plurality of reagents using the geometry of the plurality of micro-channels. Mixing the plurality of samples and the plurality of reagents produces a plurality of mixed sample droplets in the plurality of micro-channels.

In step330, one or more post-bridge detection values are received from a post-bridge detection system of the PCR system for each mixed sample droplet of the plurality of mixed sample droplets to determine if each mixed sample droplet is mixed correctly using the processor.

In step340, a thermocycler of the PCR system is instructed to maintain one or more temperatures for cycling the temperature of the plurality of mixed sample droplets in the plurality of micro-channels using the processor.

In step350, one or more endpoint detection values are received from an endpoint detection system of the PCR system for each mixed sample droplet of the plurality of mixed sample droplets to analyze the PCR experiment using the processor.

In various embodiments, a computer program product includes a non-transitory and tangible computer-readable storage medium whose contents include a program with instructions being executed on a processor so as to perform a method for high throughput PCR amplification and analysis. This method is performed by a system that includes one or more distinct software modules.

FIG. 4is a schematic diagram of a system400that includes one or more distinct software modules that perform a method for high throughput PCR amplification and analysis, in accordance with various embodiments. System400includes liquid handling module410, fluid pumping module420, post-bridge detection module430, thermocycler module440, and endpoint detection module450.

Liquid handling module410instructs a liquid handling system of a PCR system to obtain a plurality of samples and a plurality of reagents for a PCR experiment.

Fluid pumping module420instructs a fluid pumping system of the PCR system to maintain a continuous flow of a transport fluid through a plurality of micro-channels. The continuous flow allows the fluid pumping system to receive the plurality of samples and the plurality of reagents from the liquid handling system as droplets in the plurality of micro-channels. The continuous flow also allows the fluid pumping system to mix the plurality of samples and the plurality of reagents using the geometry of the plurality of micro-channels producing a plurality of mixed sample droplets in the plurality of micro-channels.

Post-bridge detection module430receives from a post-bridge detection system of the PCR system one or more post-bridge detection values for each mixed sample droplet of the plurality of mixed sample droplets to determine if each mixed sample droplet is mixed correctly.

Thermocycler module440instructs a thermocycler of the PCR system to maintain one or more temperatures for cycling the temperature of the plurality of mixed sample droplets in the plurality of micro-channels.

Endpoint detection module450receiving from an endpoint detection system of the PCR system one or more endpoint detection values for each mixed sample droplet of the plurality of mixed sample droplets to analyze the PCR experiment.

Exemplary Continuous Flow PCR System

An exemplary continuous flow PCR System is a continuous flow 96-line PCR instrument capable of sampling from master-mix, sample and primer/probes simultaneously and mixing these in a micro-channel geometry (Liquid Bridges). The mixed droplets flow downstream to a thermocycler where they are amplified. The droplets then pass a data-acquisition system where their fluorescent intensities are measured.

In order to enable system operation the following software controlled elements are present: fluid pumping system, liquid handling/plate handling system, post-bridge detection, thermocycler, endpoint detection, and ancillary equipment. The fluid pumping system includes five flow sensors, five pumps and more than 40 level sensors and valves. The liquid handling/plate handling system includes a plate stacker, a barcode reader, and a 15 axis sampling unit. The post-bridge detection includes three Basler cameras. The thermocycler includes four 24-line temperature controlled thermocyclers (TCs) each with separate denaturation blocks. The endpoint detection includes one Hamamatsu Orca camera and one laser.

FIG. 5is a schematic diagram of the software architecture for a continuous flow PCR system, in accordance with various embodiments.

FIG. 6is a flowchart showing a system initialization method, in accordance with various embodiments.

FIG. 7is a flowchart showing a method for issuing a transmission control protocol/internet protocol (TCP/IP) command, in accordance with various embodiments.

FIG. 8is a flowchart showing a first portion of a method for issuing a run command, in accordance with various embodiments.

FIG. 9is a flowchart showing a second portion of a method for issuing a run command, in accordance with various embodiments.

FIG. 10is a flowchart showing a third portion of a method for issuing a run command, in accordance with various embodiments.

FIG. 11is a flowchart showing a system shutdown method, in accordance with various embodiments.

FIG. 12is a flowchart showing a method for handling errors, in accordance with various embodiments.

Fluid Pumping System

Referring again toFIG. 2, the system200operates under the principal of continuous flow. A constant flow of oil is maintained through the thermocycler (TC line242) and this flow of oil carries mixed droplets. It is required that the flow upstream of the liquid-bridges (from sample-tips to bridges) be faster than the flow through the thermocycler in order to meet throughput demands. A draft line241is fitted to the bridge and bleeds off excess oil. The TC line242and the draft line241both operate at fixed flow rates. It is required that these lines be controlled as the addition of droplets to the lines increases the pressure drop along each line. The combined flow in the TC line242and draft Line241equals that of the master-mix, sample and primer-probe lines.

In addition the pumping system incorporates a number of subsystems for priming the system with oil and bleeding it of air.FIG. 2shows a general schematic (for a single line system) showing the TC Line242, the Draft Line241and where the hardware components are located.

If a PCR system operates under continuous flow, moving the system through air to move from well-to-well would cause air to be drawn into the system. This is avoided through the use of sheathing/flap valves. These larger bore tubes are fitted around the sampling tubes and wrap them in oil. The continuous flow of oil into the sheathing (driven by 3 independent sheathing pumps) matches (or slightly exceeds) the flow being drawn into the system tips insuring that the continuous flow lines are always wrapped in oil. Hence the tips can move freely from well to well without drawing any air into the system.

Liquid Handling/Plate Changing

FIG. 13is a schematic diagram of a flap valve opening method1300, in accordance with various embodiments. In order to facilitate the use of flap valves/sheathing (which needs to be opened before sampling can take place) the tips are mounted on a double Z-axis. The secondary axis1320is mounted on the primary axis1310. The sheathing/flap valves are mounted on primary axis1310while the tips are mounted on secondary axis1320.

In step1of method1300, in air the robotic head moves over the required wells.

In step2, primary axis1310lowers the tips (sheathing and secondary axis1320) into the oil overlay which covers the sample in each well.

In step3, secondary axis1320then extends the tips (pushing the valves open) so the tip is over the sample. Simultaneously primary axis1310rises by an equal distance. The combined effect is that secondary axis1320is stationary in space while primary axis1310moves upwards. Combined with the geometry of the flap-valves, this movement allows an extra 30 μl volume of sample be used in each (96-wellplate) well.

In step4, secondary axis1320lowers further into the well and completes opening of the flap valve. The secondary axis1320pauses until triggered to sample.

In step5, at the precise time required, secondary axis1320dips into the fluid and draws up approximately 75 nl of fluid (sample/primer-probe, master mix approx. 150 nl). The amount of fluid drawn depends on the flow-rate used and the time the tip is within the fluid.

In step6, the tip then retracts from the sample and pauses ready to sample again if required. If the next sample is needed from a neighboring well (or a plate-change) the tip retracts into the sheathing and the primary axis1310then moves the sampling head out into the air. The sheathing motion is a reverse of the unsheathing motions.

FIG. 14is a schematic diagram of a liquid/plate handling system1400, in accordance with various embodiments. In system1400, the liquid/plate handling provides movement along 15 axes. For reference, system1400is divided into three sampling systems and one plate handling system. The directions of motion of each stage are shown by arrows. Note that the sampling arm of the multi-lumen unit is shown. However, for clarity, the sampling arms of the master-mix unit and single-tip unit are rendered invisible. Additionally the master mix unit is mounted on the roof of the enclosure. The individual axes are:Single-tip SamplingX-axisY-axisPrimary Z-axis (Z1)Secondary Z-axis (Z2)Multi-lumen SamplingX-axisY-axisPrimary Z-axis (Z1)Secondary Z-axis (Z2)Rotational AxisMaster-mix SamplingX-axisPrimary Z-axis (Z1)Secondary Z-axis (Z2)Plate handlingY-axisX1-axis (Tray1—Single-tip)X2-axis (Tray2—Multi-lumen)

The single-tip system consists of 96 tips each of which can enter a single well on a 96-well or 384-well plate. Therefore system1400can sample from a 96-well plate in a single movement or a 384-well plate in four movements. The multi-lumen system consists of four bundles of 24-tips. All 24 lines in each bundle can enter a single well. Each line in the bundle is arrayed against one of the single-tip lines—meeting in a bridge and then flowing into the thermocycler. The Multi-lumen head is mounted on a rotational unit. Therefore through four rotation and dips four wells on Tray 2 (Multi-lumen side) can be arrayed against an entire 96-well plate. Similarly 16 robotic movements (four multi-lumen rotations times four single-tip movements) can permit four wells on Tray 2 be arrayed against an entire 384-well plate.

FIGS. 15A-Fis a flowchart showing a first portion of a method for plate stacking, in accordance with various embodiments.

FIGS. 16A-Bis a flowchart showing a second portion of a method for plate stacking, in accordance with various embodiments.

FIGS. 17A-Bis a flowchart showing a third portion of a method for plate stacking, in accordance with various embodiments.

FIG. 18is a flowchart showing a method for liquid handling initialization, in accordance with various embodiments.

FIGS. 19A-Bis a flowchart showing a method for liquid handling, in accordance with various embodiments.

FIG. 20is a flowchart showing a method for liquid handling shutdown, in accordance with various embodiments.

The droplet stream leaving the liquid bridges is divided into packets (based upon the time-stamp at which the robotics takes a sample). For convenience these packets are called carriages. The use of carriages—where the spacing between carriages is at least twice that between droplets—permits easier identification of individual droplets and indeed easy identification of errors in the droplet stream. For example droplet 2 of carriage 2 (with 5 droplets per carriage) may be identified more easily than droplet 12 of a continuous stream. Similarly errors can be easily identified. If only 4 droplets are present in a carriage of 5 then it is clear an error has occurred (droplet merging); if 6 are present then a droplet has not mixed or has mixed and then split into two.

FIG. 21is a state diagram showing the relationships among post-bridge methods, in accordance with various embodiments.

FIG. 22is a flowchart showing a first portion of a post-bridge initialization method, in accordance with various embodiments.

FIG. 23is a flowchart showing a second portion of a post-bridge initialization method, in accordance with various embodiments.

FIG. 24is a flowchart showing a post-bridge pre run method, in accordance with various embodiments.

FIG. 25is a flowchart showing a first portion of a post-bridge run method, in accordance with various embodiments.

FIG. 26is a flowchart showing a second portion of a post-bridge run method, in accordance with various embodiments.

FIG. 27is a flowchart showing a third portion of a post-bridge run method, in accordance with various embodiments.

FIG. 28is a flowchart showing a post-bridge run end method, in accordance with various embodiments.

FIG. 29is a flowchart showing a post-bridge shutdown method, in accordance with various embodiments.

The post-bridge detection system consists of an array of blue light emitting diodes (LEDs) illuminating the output line from the bridges (between the liquid bridges and the thermocycler). Three cameras (Basler) are used to monitor three fluorescent wavelengths excited by the blue LEDs. These components are FAM/VIC in the primer-probes, ROX in the Master-Mix and a third dye (i.e. ALEXA) added to the samples as a reference. If the detection system picks up all three wavelengths from a droplet, then this is considered a mixed and valid droplet. However in some cases the bridges will not mix a droplet correctly. This is found by determining that one or more of the components are missing from the main droplet. In the event an error occurs with a single droplet (or carriage) then this droplet (or the entire carriage) will be re-sampled.

The thermocycler includes four 24-line thermocyclers. Each block is preceded by a pre-heat block. Each block is maintained at its set-point using proportional integrated derivative (PID) control.

Endpoint Detection and Analysis

Endpoint detection consists of a free-space spectrograph system. The acquisition hardware is a Hamamatsu Orca camera. The 96 thermocycler lines are illuminated by a 488 nm laser-line. This laser-line is imaged by the spectrograph/camera and resolved into its constituent wavelengths. Appropriate wavelengths are measured according to the contents of the droplets. Droplets are identified based upon the time-stamp generated by the post-bridge detection module and raw fluorescent data is generated for droplet. Spectral compensation is then applied to compensate for dye bleed through.

The PCR instrument is driven using two different ASCII .csv files. The command file is titled in the format BARCODETRAY1_BARCODETRAY2_cmds.csv while the volume file is titled BARCODETRAY1_vols.csv. The command file contains a list of well combinations which are sampled by the instrument. The volume file contains information pertaining to the contents (volume and components) of each well on the plate. On receiving a RUN command the instrument reads the barcodes of each plate present. It searches for matching command and volume files and, if present, processes this project. Results are outputted in the form BARCODETRAY1_BARCODETRAY2_rslts.csv.

FIG. 30is a schematic diagram showing tray and position waypoints, in accordance with various embodiments. InFIG. 30liquid waypoints P1through to P6are shown. Both trays T1and T2can access all six waypoints. P1and P6are not used, for example. P2is used for barcode reading. P3is used for upstack/downstack into Hotel1on the plate-changer. P4is used similarly for Hotel2. P5is used by robots to load and unload plates.

Graphical User Interface (GUI)

A matrix of sample and reagent wells is provided to a continuous flow PCR instrument by a laboratory information management system, for example. In various embodiments, a matrix of sample and reagent wells is entered through a GUI. The GUI and the instrument interact to control the plate stacker and also to transfer files. To transfer files a file transfer protocol (FTP) setup is used. There is an FTP server that stores files and waits for clients to connect to it. The GUI acts as a client to connect to the FTP server and transfer files. The instrument can also connect to the same FTP server and transfer files.

To control the plate stacker a custom control protocol (TCP) interface is used. The instrument acts as a server and waits for the GUI to connect to it. After a connection is established predefined TCP commands are sent and received to control the instrument.

FIG. 31is a schematic diagram showing how files are transferred between a graphical user interface (GUI) and an instrument, in accordance with various embodiments. Command files and volume files can be created and modified using the GUI. These files can then be transferred to the instrument. The files are transferred using an FTP server.

FIG. 32is a flowchart showing a method for uploading a file using a file transfer protocol (FTP) server, in accordance with various embodiments. To upload a file, the GUI sends a TCP command to the instrument asking it for the address of the FTP server. Once the instrument has responded with this information, the GUI connects to the instrument and uploads a file. If the file already exists on the FTP server the user is asked if they want to keep it or overwrite it.

To download a file, the GUI sends a TCP command to the instrument asking it for the address of the FTP server. Once the instrument has responded with this information, the GUI connects to the instrument and presents a list of files available for downloading. The user selects a file, and the GUI then downloads it to a predefined location on the local computer.

The plate stacker allows the user of the instrument to load multiple plates at once and run them without having to explicitly load and run each plate combination individually. The stacker is divided into two compartments. Each compartment is loaded with plates. At run time the user tells the GUI which combinations to run. The GUI does not know which plates are in the stacker. Through a series of TCP commands instructing the instrument to transfer plates between the stacker and the instrument proper and to barcode the plates, the GUI can instruct the instrument to run all the selected combinations.

In various embodiments, a command file is a file that defines well combinations between plates, for example. An FTP server is a repository for files. The FTP server can communicate with the GUI and the instrument. A GUI sends commands to the instrument and creates files that can be stored on an FTP server. The instrument runs plates, receives commands from GUI, and interacts with an FTP server. A plate stacker is a component of the instrument that holds plates that are to be run on the instrument. TCP is a protocol that allows sending of information over a network. It is used between the GUI and the instrument. A volume file is a file that defines a plate. It contains the plate barcode, plate type, and volumes of wells.

Endpoint Detection System

In order to maintain the high throughput of a continuous flow PCR system, the PCR system needs to be able to detect fluorescence in two or more micro-channels at the same time. Measuring fluorescence across two or more micro-channels imposes a number of limitations on an endpoint detection system.

For example, as the number of number of micro-channels is increased, the field of view of the detector also needs to increase. These micro-channels can be closely bundled or aligned together in an array of transparent micro-channels or tubes. However, a wall of some thickness has to be maintained between tubes to prevent crosstalk between adjacent micro-channels. As a result, the field of view of the detector is a function of the tube diameter and tube array wall thickness. In order to maintain a high fluorescence collection efficiency from the tubes on the edges of the tube array, an increased beam length can be used. Increasing the beam length from the tube array to the detector increases the overall physical size of the endpoint detection system, however.

Also, a laser is a typical illumination source for fluorescence measurements. The power distribution of a laser beam is highly non-uniform. This power distribution generally follows a Gaussian distribution and drops exponentially off-axis. However, an amplification system of a continuous flow PCR system needs an illumination source with a uniform power distribution to illuminate the entire width of the tube array.

Finally, because the flow of samples is continuous in the tube array, the PCR system has to be able to detect spectral information from two or more micro-channels in a single time step. However, in order to assign that spectral information to the correct sample, the particular tube emitting that spectral information needs to be located in the tube array. As result, the endpoint detection system needs to provide spatial information in addition to spectral information.

FIG. 33is a schematic diagram of a side view of a system3300for detecting spectral and spatial information in a continuous flow PCR system, in accordance with various embodiments. System3300includes laser3310, line generator3320, tube array3330, imaging lens3340, spectrograph3350, and imager3360. Laser3310emits incident beam of electromagnetic radiation3311.

Line generator3320receives incident beam3311from laser3310. Line generator3320transforms incident beam3311into incident line of electromagnetic radiation3321. On other words, line generator3320converts the power distribution of incident beam3311from a non-uniform distribution to a uniform distribution. Line generator3320is a Powell lens, for example. In various embodiments, line generator3320is a diffractive line generator.

Tube array3330receives incident line3321from line generator3320. Tube array3330includes one or more transparent tubes in fluid communication with one or more micro-channels of a PCR system. In various embodiments, one or more optical elements3322are placed between line generator3320and tube array3320to steer incident line3321from line generator3320to tube array3330. As shown inFIG. 33, one or more optical elements3322allow system3300to be package in an overall smaller volume, for example. In various embodiments, mirror3325is also placed between line generator3320and tube array3330to steer incident line3321from line generator3320to tube array3330. Mirror3325allows tube array3330to be positioned horizontally in system3300, for example.

Imaging lens3340receives reflected electromagnetic radiation3331from tube array3330and focuses reflected electromagnetic radiation3331. In various embodiments, one or more optical elements (not shown) are placed between tube array3330and imaging lens3340to steer reflected electromagnetic radiation3331from tube array3330to imaging lens3340. In various embodiments, mirror3325is placed between tube array3330and imaging lens3340to steer reflected electromagnetic radiation3331from tube array3330to imaging lens3340. Imaging lens3340is a wide-iris lens with a variable aperture, for example. In various embodiments, imaging lens3340includes one or more optical filters (not shown). The one or more optical filters remove reflection of incident line3321from reflected electromagnetic radiation3331, for example.

Spectrograph3350receives the focused reflected electromagnetic radiation (not shown) from the imaging lens3340. Spectrograph3350detects a spectral intensity from the focused reflected electromagnetic radiation. Spectrograph3350can detect spectral wavelengths between 400 and 800 nanometers, for example.

Imager3360receives the focused reflected electromagnetic radiation from imaging lens3340. Imager3360detects a location of the spectral intensity. Imager3360is a CCD camera, for example.

In various embodiments, system3300also includes a processor (not shown). The processor receives the spectral intensity from spectrograph3350and receives the location from imager3360. The processor determines an intensity value for a sample moving through tube array3330from the spectral intensity and the location.

FIG. 34is a schematic diagram of a top view of a system3400for detecting spectral and spatial information in a continuous flow PCR system, in accordance with various embodiments.

FIG. 35is a schematic diagram of a three-dimensional view of a tube array plate, in accordance with various embodiments.

FIG. 36is a schematic diagram of a top view of a tube array plate, in accordance with various embodiments.

FIG. 37is a schematic diagram of a side view of a tube array plate, in accordance with various embodiments.

FIG. 38is a flowchart showing a method3800for detecting spectral and spatial information in a continuous PCR system, in accordance with various embodiments.

In step3810of method3800, an incident beam of electromagnetic radiation is emitted using a laser.

In step3820, the incident beam is received from the laser and the incident beam is transformed into an incident line of electromagnetic radiation using a line generator.

In step3830, the incident line is received from the line generator using a tube array that includes one or more transparent tubes in fluid communication with one or more micro-channels of a PCR system.

In step3840, reflected electromagnetic radiation is received from the tube array and the reflected electromagnetic radiation is focused using an imaging lens.

In step3850, the focused reflected electromagnetic radiation is received from the imaging lens and a spectral intensity is detected from the focused reflected electromagnetic radiation using a spectrograph.

In step3860, the focused reflected electromagnetic radiation is received from the imaging lens and a location of the spectral intensity is detected using an imager.