WO2016065299A2

WO2016065299A2 - Ultrasonics for microfluidic sample preparation

Info

Publication number: WO2016065299A2
Application number: PCT/US2015/057184
Authority: WO
Inventors: Mark W. Eshoo; John Picuri; Alice T. HANG; Heather L. Smith; Bernadet Meijering; Rene KRAGT; M. Bruyninckx; I. Dobbelaer
Original assignee: Eshoo Mark W; John Picuri; Hang Alice T; Smith Heather L; Bernadet Meijering; Kragt Rene; Bruyninckx M; Dobbelaer I
Priority date: 2014-10-24
Filing date: 2015-10-23
Publication date: 2016-04-28
Also published as: WO2016065299A3

Abstract

Provided herein is technology relating to microfluidics and particularly, but not exclusively, to devices, apparatuses, methods, and systems for preparing and analyzing biomolecules using an ultrasonic device.

Description

ULTRASONICS FOR MICROFLUIDIC SAMPLE PREPARATION

CROSS-REFERENCE TO RELATED APPLICATIONS

The present Application claims priority to U.S. Provisional Application Serial Number 62/068,406 filed October 24, 2014, the entirety of which is incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant number

HDTRAl-lO-c-0080 awarded by the Defense Threat Reduction Agency. The government has certain rights in the invention.

FIELD

Provided herein is technology relating to microfluidics and particularly, but not exclusively, to devices, apparatuses, methods, and systems for processing samples comprising biomolecules using an ultrasonic device.

BACKGROUND

The use of molecular diagnostics has expanded greatly since its inception in the early 1980s, particularly as a means to detect bacteria, viruses, and for molecular diagnostics in pharmacogenomics, companion diagnostics, and other personalized medicine applications. In general, molecular diagnostic technologies are based on analysis of one or more isolated nucleic acids prepared from a biological sample.

Traditionally, preparation of nucleic acid from a biological sample is labor-intensive, time-intensive, and traditionally requires varied and expensive laboratory equipment. Accordingly, a need exists for integrated and automated preparation of nucleic acid on a small scale.

SUMMARY

Accordingly, provided herein is technology related to isolating nucleic acids from a biological sample. In particular, the technology is related to lysing cells and fragmenting the released nucleic acids using a temperature-controlled ultrasonic device adaptable for inclusion in a microfluidic apparatus.

In general, studying or analyzing the sequence and biology of DNA or RNA from a sample usually involves extracting, purifying, or isolating the nucleic acids from the rest of the clinical or biological sample (e.g., from other cellular components such as lipids, carbohydrates, proteins, etc.). Existing methods presently performed by those familiar with the art include a variety of chemical, biological (e.g., enzymatic), and physical methods that vary depending upon application and sample type. For instance, methodologies typically begin with cell disruption or cell lysis to release nucleic acids. This is commonly achieved by physical (e.g., mechanical) lysis (such as grinding (e.g., grinding tissue in liquid nitrogen), bead beating, sonicating, etc.), biological (e.g., enzymatic) lysis, and/or chemical lysis (e.g., adding a chaotropic salt (e.g., guanidinium thiocyanate) to the sample). Lipids (e.g., from membranes and other sources) are usually removed by adding a detergent and proteins usually removed by adding a protease (e.g., proteinase K). Then, nucleic acids are typically obtained from the lysate by extraction (e.g., phenol/chloroform extraction) or using solid phase technologies (e.g., binding to silica in the presence of high concentrations of chaotropic salts).

Further, analysis of nucleic acids often involves generating fragments having a size within a defined size range from the larger nucleic acids. For example, fragmented nucleic acid (e.g., DNA) is increasingly being used in the preparation of sequencing libraries (e.g., for next-generation sequencing (e.g., NGS)).

In accordance with the technology describe herein, ultrasound provides a particular technology for lysing cells and/or fragmenting nucleic acids. Ultrasonic devices create mechanical stress to rupture cell walls (see, e.g., U.S. Pat. No. 4,874, 137). The ultrasound waves expand and contract the liquid in a sample. During expansion, molecules are pulled away from one another and cavities or bubbles are formed in a process called cavitation. After formation, a bubble continues to absorb energy until it can no longer sustain itself and then it implodes, producing intense focused shearing forces that rupture cell walls and thus effect cell lysis. In addition, ultrasound in the kilohertz and megahertz ranges finds use in fragmenting DNA (see, e.g., Mann and Krull (2004) "The application of ultrasound as a rapid method to provide DNA fragments suitable for detection by DNA biosensors" Biosensors and Bioelectronics 20: 945-955; Rageh et al. (2009) "Effect of high power ultrasound on aqueous solution of DNA" International Journal of Physical Sciences 4- 63-68; and U.S. Pat. No. 6,719,449). Some parameters that are associated with the use of ultrasound to prepare fragmented DNA include selecting the size of the sample, the periodicity of activation of the ultrasound waves and duty cycle, and the total duration of treatment.

Some amount of the vibrational energy introduced into the sample is converted to heat. Thus, treatment of samples with ultrasonics heats the samples and can damage biological components (e.g., DNA) present in the samples. Accordingly, the technology provided herein is related to controlling the temperature of samples treated with ultrasonic devices. In some embodiments, a thermoelectric device (e.g., a Peltier device) provides for temperature control of samples, e.g., by providing a heat sink to remove heat energy from samples and/or ultrasonic devices used to treat samples.

Accordingly, provided herein is a device for producing nucleic acid fragments from a biological sample comprising nucleic acids. In some embodiments, the device comprises a non-focusing ultrasonic transducer comprising a hole and a thermoelectric component attached to the non-focusing ultrasonic transducer. In some embodiments, the hole provides for coupling of the transducer to a sample chamber (e.g., comprising a sample) and/or minimizes or eliminates the formation of bubbles in the sample.

In some embodiments, the ultrasonic transducer is a transducer that produces frequencies in the MHz range (e.g., 1 to 1000 MHz, e.g., 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 MHz, e.g., in some embodiments from 1 to 10, e.g., 1 to 5, e.g., 1 to 2, e.g., 1.7 MHz). In some embodiments, the device further comprises an insulating ring surrounding the ultrasonic transducer. In some embodiments, the insulating ring comprises a heat resistant material. For example, in some embodiments the insulating ring comprises a resin-reinforced, glass-reinforced, or polymer-reinforced laminate material, e.g., a glass-reinforced epoxy such as an FR4 material. In some embodiments, the thermoelectric component is a Peltier type thermoelectric component. In some embodiments, the thermoelectric component comprises a coating (e.g., a polymer coating such as, e.g., a PTFE coating, a

perfluoroalkoxy (PFA) coating, a fluorinated ethylene propylene (FEP) coating, a polychlorotrifluoroethylene coating, a tetrafluorethylene-perfluoropropylene coating, a polyether ether ketone (PEEK) coating, or a nylon coating) to minimize or eliminate adherence of the thermoelectric component to other components, modules, etc. The technology is not limited in the type of ultrasonic transducer that is used to produce sonic energy and provide sonic energy into a sample. Exemplary types of ultrasonic transducers include a piezoelectric transducer, an electromagnetic transducer, and a capacitive transducer. In some embodiments the device further comprises a heat sink, e.g., in thermal communication with the thermoelectric component, e.g., to remove thermal energy from the device. Some embodiments of the device comprise a

temperature sensor.

In some embodiments, the thermoelectric component provides for the

maintenance of a temperature, e.g., of the device or of the sample. For instance, in some embodiments the thermoelectric device maintains the sample at a temperature at which a nucleic acid is not degraded or denatured. For example, in some embodiments, the thermoelectric component maintains the temperature of a sample below 99°C, below 95°C, below 90°C, below 85°C, below 80°C, below 75°C, or below 70°C. The device produces nucleic acid fragments over a range of sizes that are smaller than the size of the nucleic acids in the input biological sample. For instance, embodiments of the technology produce nucleic acid fragments that are appropriate for introduction into a NGS workflow, e.g., to produce a sequencing library for input to a NGS sequencing apparatus. The technology is not limited in the specific type of NGS platform for which nucleic acid fragments are produced. For instance, embodiments of the technology generate nucleic acid fragments having a size that is approximately 100 bp to 5000 bp (e.g., 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, 550 bp, 600 bp, 650 bp, 700 bp, 750 bp, 800 bp, 850 bp, 900 bp, 950 bp, 1000 bp, 1050 bp, 1100 bp, 1150 bp, 1200 bp, 1250 bp, 1300 bp, 1350 bp, 1400 bp, 1450 bp, 1500 bp, 1550 bp, 1600 bp, 1650 bp, 1700 bp, 1750 bp, 1800 bp, 1850 bp, 1900 bp, 1950 bp, 2000 bp, 2050 bp, 2100 bp, 2150 bp, 2200 bp, 2250 bp, 2300 bp, 2350 bp, 2400 bp, 2450 bp, 2500 bp, 2550 bp, 2600 bp, 2650 bp, 2700 bp, 2750 bp, 2800 bp, 2850 bp, 2900 bp, 2950 bp, 3000 bp, 3050 bp, 3100 bp, 3150 bp, 3200 bp, 3250 bp, 3300 bp, 3350 bp, 3400 bp, 3450 bp, 3500 bp, 3550 bp, 3600 bp, 3650 bp, 3700 bp, 3750 bp, 3800 bp, 3850 bp, 3900 bp, 3950 bp, 4000 bp, 4050 bp, 4100 bp, 4150 bp, 4200 bp, 4250 bp, 4300 bp, 4350 bp, 4400 bp, 4450 bp, 4500 bp, 4550 bp, 4600 bp, 4650 bp, 4700 bp, 4750 bp, 4800 bp, 4850 bp, 4900 bp, 4950 bp, or 5000 bp). In some embodiments, the device produces nucleic acid fragments of 250 to 1000 base pairs; in some embodiments, the device produces nucleic acid fragments of 1000 to 3000 base pairs.

In some embodiments, the technology provides nucleic acid fragments (e.g., for

NGS technologies) that are in the ranges of 1 kbp to 5 kbp, 10 kbp, 15 kbp, 20 kbp, 30 kbp, 40 kbp, or up to 50 kbp (e.g., approximately 1000 bp; 2000 bp; 3000 bp; 4000 bp; 5000 bp; 6000 bp; 7000 bp; 8000 bp; 9000 bp; 10,000 bp; 11,000 bp; 12,000 bp; 13,000 bp; 14,000 bp; 15,000 bp; 16,000 bp; 17,000 bp; 18,000 bp; 19,000 bp; 20,000 bp; 25,000 bp; 30,000 bp; 35,000 bp; 40,000 bp; 45,000 bp; or 50,000 bp).

Embodiments of the technology couple an ultrasonic device to a vessel containing a sample without the use of a coupling agent such as a coupling gel or coupling liquid. Accordingly, in some embodiments the device is adapted for dry coupling to a vessel containing the sample. Accordingly, in particular embodiments the technology provides a device comprising a non-focusing piezoelectric transducer comprising a hole (e.g., to minimize or eliminate the formation of bubbles in the sample and/or to provide coupling by vacuum); a Peltier type thermoelectric component attached to the non-focusing piezoelectric transducer and comprising a coating (e.g., a polymer coating such as, e.g., a PTFE coating, a perfluoroalkoxy (PFA) coating, a fluorinated ethylene propylene (FEP) coating, a polychlorotrifluoroethylene coating, a tetrafluorethylene-perfluoropropylene coating, a polyether ether ketone (PEEK) coating, or a nylon coating); a temperature sensor; and an insulating ring comprising a heat resistant material (e.g., comprising a re sin- reinforced, glass-reinforced, or polymer-reinforced laminate material, e.g., a glass- reinforced epoxy such as an FR4 material), wherein the device is adapted to be dry coupled to a vessel (e.g., a microfluidic cartridge) containing a sample.

Further embodiments of the technology provide an apparatus for producing nucleic acid fragments from a biological sample comprising nucleic acid. In some embodiments, the apparatus comprises a plurality of devices as described above (e.g., comprising a non-focusing ultrasonic transducer comprising a hole and a thermoelectric component attached to the non-focusing ultrasonic transducer). In some embodiments, the apparatus comprises a first device as described herein that is configured to lyse cells and a second device as described herein that is configured to fragment nucleic acids. The apparatus comprises in some embodiments an interface (e.g., a slot, bay, or other opening or orifice) to accept a microfluidic cartridge. In some embodiments, a

microfluidic cartridge comprises a cell lysis chamber and/or a nucleic acid fragmentation chamber (e.g., in some embodiments, a cell lysis chamber and/or a nucleic acid fragmentation chamber comprising a vent (e.g., to release pressure), e.g., a vent comprising a HEPA filter). Accordingly, in some embodiments the apparatus comprises a microfluidic cartridge comprising a cell lysis chamber and a nucleic acid fragmentation chamber (e.g., when the microfluidic cartridge is inserted into the interface of the apparatus configured to accept a microfluidic cartridge). In some embodiments, the apparatus comprises a feature to maintain a firm connection between the ultrasonic device and a vessel comprising a sample. For instance, in some embodiments the apparatus comprises a spring or vacuum to a hold a device tightly against a cell lysis chamber and in some embodiments the apparatus comprises a spring or a vacuum to hold a device tightly against a nucleic acid fragmentation chamber. The apparatus lyses cells and produces fragments of nucleic acids; accordingly, in some embodiments the apparatus comprises a sensor to monitor cell lysis and/or a sensor to monitor nucleic acid fragmentation. Examples of sensors include a sensor to monitor scattered light, a sensor to monitor fluorescence emission, a sensor to monitor ultraviolet and/or visible light, a sensor to monitor viscosity, a sensor to monitor an ion concentration (e.g., a sensor to monitor calcium ions, sodium ions, pH, etc.), a sensor to measure conductivity.

Accordingly, the technology is related to an apparatus comprising a device for lysing cells, a device for fragmenting nucleic acids, one or more sensors to monitor cell lysis and/or nucleic acid fragmentation, and an interface configured or adapted to accept a microfluidic cartridge, wherein one or more of the devices comprise(s) a non-focusing piezoelectric transducer comprising a hole (e.g., to minimize or eliminate the formation of bubbles in the sample and/or to provide coupling by vacuum); a Peltier type thermoelectric component attached to the non-focusing piezoelectric transducer and comprising a coating (e.g., a polymer coating such as, e.g., a PTFE coating, a

perfluoroalkoxy (PFA) coating, a fluorinated ethylene propylene (FEP) coating, a polychlorotrifluoroethylene coating, a tetrafluorethylene-perfluoropropylene coating, a polyether ether ketone (PEEK) coating, or a nylon coating); a temperature sensor; and an insulating ring comprising a heat resistant material (e.g., an insulating ring comprising a resin-reinforced, glass-reinforced, or polymer-reinforced laminate material, e.g., a glass-reinforced epoxy such as an FR4 material) and wherein one or more of the device(s) is/are adapted to be dry coupled (e.g., by a vacuum or a spring) to a microfluidic cartridge containing a sample. The technology is not limited in the frequency produced by the transducer. For example, in some embodiments the transducer produces frequencies in the MHz range (e.g., 1 to 1000 MHz, e.g., 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 MHz, e.g., in some embodiments from 1 to 10, e.g., 1 to 5, e.g., 1 to 2, e.g., 1.7 MHz).

Further embodiments of the technology are related to a method for producing nucleic acid fragments from a biological sample comprising nucleic acid. For example, some embodiments of the method comprise providing an ultrasonic device as described above or an apparatus as described above (e.g., an apparatus comprising a plurality of devices as described above), lysing cells in the biological sample using ultrasonic energy (e.g., produced from an ultrasonic device) to produce a lysate; and fragmenting nucleic acids in the lysate using ultrasonic energy to produce an output sample comprising fragmented nucleic acids. In some embodiments, the methods comprise monitoring the temperature of the biological sample, the lysate, and/or the output sample and some embodiments comprise providing electric current and/or electric voltage to the device or apparatus when the temperature of the biological sample, the lysate, and/or the output sample is below a maximum temperature (e.g., a temperature below 99°C, e.g., below 95°C, below 90°C, below 85°C, below 80°C, e.g., below 71, 72, 73, 74, 75, 76, 77, 78, or 79, e.g., 75°C). Embodiments comprise providing electric current and/or electric voltage to the device for 10 minutes or less (e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1 minutes). In some embodiments, embodiments of the method comprise providing a current of 0.1 to 0.5 amperes to an ultrasonic transducer (e.g., providing 0.1, 0.2, 0.3, 0.4, or 0.5 amperes, e.g., 0.3 amperes). In some embodiments, the ultrasonic transducer is a transducer that produces frequencies in the MHz range (e.g., 1 to 1000 MHz, e.g., 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 MHz, e.g., in some embodiments from 1 to 10, e.g., 1 to 5, e.g., 1 to 2, e.g., 1.7 MHz, approximately 20 MHz, etc.) Embodiments provide a method further comprising cooling the device, apparatus, or sample with a thermoelectric component (e.g., a Peltier type thermoelectric

component). The technology finds use in preparing sequencing libraries, e.g., for input to a NGS workflow. Accordingly, embodiments of the method comprise outputting the output sample to a NGS apparatus. Some embodiments of methods comprises dry coupling the device or apparatus to a sample chamber holding the biological sample, e.g., some embodiments provide holding the device or apparatus to a sample chamber using a spring or a vacuum.

The methods are related to processing samples provided in a microfluidic cartridge; thus, in some embodiments the methods comprise providing a microfluidic cartridge comprising the biological sample and, in some embodiments, further comprise inserting a microfluidic cartridge comprising the biological sample into an interface of the apparatus adapted to receive the microfluidic cartridge. In some embodiments, methods comprise providing a microfluidic cartridge comprising the biological sample and beads to cavitate and/or mix the sample (e.g., providing a microfluidic cartridge comprising the biological sample and grinding beads such as, e.g., hardened steel beads, stainless steel beads, tungsten carbide beads, agate beads, zirconium oxide beads, PTFE beads, yttrium stabilized zircon beads, glass beads, or silica beads, e.g., having a diameter of approximately 1 to 5 mm, e.g., 2 mm). In some embodiments, the methods comprise providing a microfluidic cartridge comprising one or more sample chambers connected to a depressurization chamber by a vented conduit comprising an aerosol filter (e.g., a HEPA filter). Embodiments of the methods produce an output sample comprising nucleic acid fragments of 250 to 1000 base pairs, comprising nucleic acid fragments of 1000 to 3000 base pairs, comprising 1 kbp to 50 kbp (e.g., approximately 1000 bp; 2000 bp; 3000 bp; 4000 bp; 5000 bp; 6000 bp; 7000 bp; 8000 bp; 9000 bp; 10,000 bp; 11,000 bp; 12,000 bp; 13,000 bp; 14,000 bp; 15,000 bp; 16,000 bp; 17,000 bp; 18,000 bp; 19,000 bp; 20,000 bp; 30,000 bp; 40,000 bp; or 50,000 bp), etc.

Accordingly, particular method embodiments according to the technology comprise^ l) providing a microfluidic cartridge comprising one or more sample chambers (e.g., a cell lysis chamber and/or a nucleic acid fragmentation chamber, which, in some embodiments comprise(s) a vent, e.g., a vent comprising a HEPA filter) comprising a biological sample and beads to cavitate and/or mix the sample (e.g., grinding beads such as, e.g., hardened steel beads, stainless steel beads, tungsten carbide beads, agate beads, zirconium oxide beads, PTFE beads, yttrium stabilized zircon beads, glass beads, or silica beads, e.g., having a diameter of approximately 1 to 5 mm, e.g., 2 mm); 2) providing an apparatus comprising a device for lysing cells, a device for fragmenting nucleic acids, one or more sensors to monitor cell lysis and/or nucleic acid fragmentation, and an interface configured or adapted to accept the microfluidic cartridge, wherein one or more of the devices comprise(s) a non-focusing piezoelectric transducer comprising a hole; a Peltier type thermoelectric component attached to the non-focusing piezoelectric transducer and comprising a coating (e.g., a polymer coating such as, e.g., a PTFE coating, a perfluoroalkoxy (PFA) coating, a fluorinated ethylene propylene (FEP) coating, a polychlorotrifluoroethylene coating, a tetrafluorethylene-perfluoropropylene coating, a polyether ether ketone (PEEK) coating, or a nylon coating); a temperature sensor; and an insulating ring comprising a resin-reinforced, glass -reinforced, or polymer-reinforced laminate material, e.g., a glass-reinforced epoxy such as an FR4 material; 3) dry-coupling (e.g., by a vacuum or a spring) one or more devices to the microfluidic cartridge; 4) lysing cells in the biological sample using ultrasonic energy to produce a lysate; 5) fragmenting nucleic acids in the lysate using ultrasonic energy to produce an output sample comprising fragmented nucleic acids; and 6) outputting the sample comprising fragmented nucleic acids, e.g., to a NGS sequencer. In some embodiments the methods further comprise providing a current of approximately 0.3 amperes to the one or more ultrasonic devices for less than approximately 10 minutes, monitoring the temperature of the sample, and maintaining the temperature of the sample to be below 99°C, 95°C, 90°C, 85°C, 80°C, or 75°C (e.g., by cooling the sample with the thermoelectric component).

Even more embodiments of the technology are related to a system for producing nucleic acid fragments from a biological sample comprising nucleic acid. For example embodiments of systems comprise a microfluidic cartridge comprising a sample chamber to hold a biological sample and an apparatus comprising a non-focusing ultrasonic transducer comprising a hole to lyse cells and/or to fragment nucleic acids; and a thermoelectric component attached to the non-focusing ultrasonic transducer to cool the ultrasonic transducer and/or to cool the biological sample. Further embodiments comprise a temperature sensor to monitor the temperature of the ultrasonic transducer and/or biological sample. In addition, embodiments of systems comprise a

microprocessor to receive temperature data from a temperature sensor and to provide an electric current or voltage to the ultrasonic transducer. In particular embodiments, the system comprises a high-speed buffered digital input/output to provide a low ripple DC voltage to the thermoelectric component. System embodiments comprising microfluidic cartridges comprise, in some embodiments, a microfluidic cartridge that comprises one or more sample chambers connected to a depressurization chamber by a vented conduit comprising an aerosol filter (e.g., a HEPA filter). In some embodiments, the microfluidic cartridge comprises grinding beads such as, e.g., hardened steel beads, stainless steel beads, tungsten carbide beads, agate beads, zirconium oxide beads, PTFE beads, yttrium stabilized zircon beads, glass beads, or silica beads, e.g., having a diameter of approximately 1 to 5 mm, e.g., 2 mm. Some embodiments provide that the microfluidic cartridge is dry coupled to the ultrasonic transducer.

Accordingly, particular embodiments of systems comprise^ l) a microfluidic cartridge comprising one or more sample chambers (e.g., a cell lysis chamber and/or a nucleic acid fragmentation chamber, e.g., comprising a vent, e.g., comprising a vent comprising a HEPA filter) comprising a biological sample and beads to cavitate and/or mix the sample (e.g., grinding beads such as, e.g., hardened steel beads, stainless steel beads, tungsten carbide beads, agate beads, zirconium oxide beads, PTFE beads, yttrium stabilized zircon beads, glass beads, or silica beads, e.g., having a diameter of approximately 1 to 5 mm, e.g., 2 mm)); 2) an apparatus comprising a device for lysing cells, a device for fragmenting nucleic acids, one or more sensors to monitor cell lysis and/or nucleic acid fragmentation, and an interface configured or adapted to accept the microfluidic cartridge, wherein one or more of the devices comprise(s) a non-focusing piezoelectric transducer comprising a hole; a Peltier type thermoelectric component attached to the non-focusing piezoelectric transducer and comprising a coating (e.g., a polymer coating such as, e.g., a PTFE coating, a perfluoroalkoxy (PFA) coating, a fluorinated ethylene propylene (FEP) coating, a polychlorotrifluoroethylene coating, a tetrafluorethylene-perfluoropropylene coating, a polyether ether ketone (PEEK) coating, or a nylon coating); a temperature sensor; and an insulating ring comprising a heat resistant material such as a resin-reinforced, glass-reinforced, or polymer-reinforced laminate material, e.g., a glass-reinforced epoxy such as an FR4 material.; 3) a high^¬ speed buffered digital input/output to provide a low ripple DC voltage to the

thermoelectric component; and 4) a microprocessor to receive temperature data from a temperature sensor and to provide an electric current or voltage to the ultrasonic transducer.

These systems, in some embodiments, are integrated into larger systems, e.g., systems for sequencing a nucleic acid. Thus, further embodiments provide a system for sequencing a nucleic acid comprising embodiments of the above described systems and a NGS sequencing apparatus.

In some embodiments, various components that are described above are integrated into one system. For example, some embodiments provide an integrated system (e.g., a microfluidic cartridge) comprising an ultrasonic component (e.g., for cell lysis and/or for nucleic acid fragmentation), a thermoelectric component, a sample chamber (e.g., comprising a vent, e.g., a vent comprising a HEPA filter), a sequencing component, an amplification component, a nucleic acid extraction and/or purification component, and a microcontroller or microprocessor. Some integrated systems comprise a power source, e.g., a battery, fuel cell, or other current and/or voltage source. Some integrated systems provide a communications output (e.g., a wired or wireless network connection) to communicate data (e.g., nucleic acid sequence data) to another networked computer. Accordingly, in some embodiments the technology comprises an integrated system comprising an input to provide a biological sample and all components, modules, technologies, reagents, etc. to prepare and sequence nucleic acids present in the biological sample and communicate one or more nucleic acid sequences to another networked computer.

Additional embodiments will be apparent to persons skilled in the relevant art based on the teachings contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the present technology will become better understood with regard to the following drawings^

Figure 1 is a series of drawings of an embodiment of an ultrasonic device described herein. Figure 1A shows a top perspective view of an embodiment of an ultrasonic device described herein. Figure IB shows a bottom view of the embodiment of the ultrasonic device shown in Figure 1A. Figure 1C shows a cross-section view of the embodiment of the ultrasonic device shown in Figure 1A cut along the line A-A shown in Figure IB. Figure 1C indicates the following components of the embodiment of the ultrasonic device^ piezoelectric component 110, thermoeletric component 120,

temperature sensor(s) 130, seal and/or insulating ring 140, thermal interface 150 for thermal communication to a heat sink, and wiring or cables 160. Figure ID is a side view of the ultrasonic device shown in Figure 1A.

Figure 2 shows two plots of sample temperature and piezoelectric transducer temperature versus fragmentation time for two ultrasonic devices.

Figure 3 is a drawing of an embodiment of an ultrasonic device comprising a piezoelectric transducer comprising a hole.

Figure 4 shows the size distributions for input nucleic acids ("Input WGA"), nucleic acid fragments produced with an ultrasonic device comprising a piezoelectric transducer that does not comprise a hole ("3x3 min US"), and nucleic acid fragments produced with an ultrasonic device comprising a piezoelectric transducer comprising a hole ("9 min US piezo + hole").

Figure 5 is a plot showing the peak in the fragment size distribution as a function of fragmentation time.

Figure 6 is a plot showing the peak in the fragment size distribution as a function of the current provided to the piezoelectric transducer.

Figure 7 is a plot showing the temperature of the sample and the piezoelectric transducer during a fragmentation time of 10 minutes supplying a current of 0.3 amperes to the piezoelectric transducer.

Figure 8 is a bar plot showing the effect of duty cycle on the peak of the fragment size distribution. Duty cycle is adjusted by adjusting the maximum set temperature at which the device is run before turning off the power to initiate a cooling interval.

Figure 9 is an infrared image showing the thermal isolation of the ultrasonic device provided by the insulating ring in particular embodiments of the technology provided herein. Dark grey and black indicates high temperatures (e.g., near 70 to 80°C) and light grey and white indicates low temperatures (e.g., near 20 to 25°C). The grey ring separating the black and white colors is the insulating ring.

It is to be understood that the figures are not necessarily drawn to scale, nor are the objects in the figures necessarily drawn to scale in relationship to one another. The figures are depictions that are intended to bring clarity and understanding to various embodiments of apparatuses, systems, and methods disclosed herein. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. Moreover, it should be appreciated that the drawings are not intended to limit the scope of the present teachings in any way.

DETAILED DESCRIPTION

Provided herein is technology is related to lysing cells and fragmenting the released nucleic acids using a temperature-controlled ultrasonic device adaptable for inclusion in a microfluidic apparatus

In this detailed description of the various embodiments, for purposes of explanation, numerous specific details are set forth to provide a thorough understanding of the embodiments disclosed. One skilled in the art will appreciate, however, that these various embodiments may be practiced with or without these specific details. In other instances, structures and devices are shown in block diagram form. Furthermore, one skilled in the art can readily appreciate that the specific sequences in which methods are presented and performed are illustrative and it is contemplated that the sequences can be varied and still remain within the spirit and scope of the various embodiments disclosed herein. The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way.

All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which the various embodiments described herein belongs. When definitions of terms in incorporated references appear to differ from the definitions provided in the present teachings, the definition provided in the present teachings shall control.

Definitions

To facilitate an understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase "in one embodiment" as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase "in another embodiment" as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

In addition, as used herein, the term "or" is an inclusive "or" operator and is equivalent to the term "and/or" unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of "a", "an", and "the" include plural references. The meaning of "in" includes "in" and "on."

The term "module," as used herein, refers to a device, component, or apparatus that includes one or more parts or independent units that are configured to be part of a larger device or apparatus. In some cases, a module works independently from another module. In other cases, a module works in conjunction with other modules (e.g., modules within modules) to perform one or more tasks, such as processing a biological sample.

The term "cells," as used in the context of biological samples, encompasses samples that are generally of similar sizes to individual cells, including but not limited to vesicles (such as liposomes), cells, virions, and substances bound to small particles such as beads, nanoparticles, or microspheres. Characteristics of cells include, but are not limited to, size; shape; temporal and dynamic changes such as cell movement or multiplication; granularity; whether the cell membrane is intact; internal cell contents, including but not limited to, protein content, protein modifications, nucleic acid content, nucleic acid modifications, organelle content, nucleus structure, nucleus content, internal cell structure, contents of internal vesicles, ion concentrations, and presence of other small molecules such as steroids or drugs; and cell surface (both cellular membrane and cell wall) markers including proteins, lipids, carbohydrates, and modifications thereof.

As used herein, the term "ripple" when used in reference to electrical current such as a direct current (DC) refers to a small residual periodic variation of the direct current (DC) output of a power supply that has been derived from an alternating current (AC) source. This ripple is due to incomplete suppression of the alternating waveform within the power supply.

As used herein, the term "nucleic acid" means single-stranded and double- stranded polymers of nucleotide monomers, including, but not limited to, 2'- deoxyribonucleotides and ribonucleotides (RNA) linked by internucleotide

phosphodiester bond linkages, e.g. 3'-5' and 2'-5', inverted linkages, e.g. 3'-3' and 5'-5', branched structures, or analog nucleic acids. Nucleic acids can be natural nucleic acids, artificial nucleic acids, analogs thereof, or combinations thereof. Nucleic acids have associated counter ions, such as H⁺, NH⁴⁺, trialkylammonium, Mg²⁺, Na⁺, and the like. A nucleic acid can be composed entirely of deoxyribonucleotides, entirely of

ribonucleotides, or chimeric mixtures thereof. Nucleic acid can be comprised of nucleobase and sugar analogs. Nucleic acid typically range in size from a few monomeric units, e.g. 5-40, when they are more commonly frequently referred to in the art as oligonucleotides, to several thousands of monomeric nucleotide units. Unless denoted otherwise, whenever a nucleic acid sequence is represented, it will be understood that the nucleotides are in 5' to 3' order from left to right and that "A" denotes

deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine.

Nucleic acids are said to have "5' ends" and "3' ends" because mononucleotides are reacted to make nucleic acids in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of a nucleic acid is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger nucleic acid, also can be said to have 5' and 3' ends.

As used herein, "purified nucleic acid" denotes a genomic polynucleotide without or associated with a reduced amount of cellular material. For example, a sample from a cell, where the polynucleotide is fragmented into acceptable sizes to serve as nucleic acid fragments. The options and variations of purification of the genomic polynucleotide are broadly known to one skilled in the art of cellular lysis and vary on the cellular material and inhibitors that can be contained in that material.

As used herein, the term "microarray" encompasses an arrangement of polynucleotides present on a solid support or in an arrangement of vessels. Certain array formats are referred to as a "chip" or "biochip" (M. Schena, Ed. Microarray Biochip Technology, BioTechnique Books, Eaton Publishing, Natick, Mass. (2000)). An array can comprise a low-density number of addressable locations, e.g. 1 to about 12, medium- density, e.g. about a hundred or more locations, or a high-density number, e.g. a thousand or more. Typically, the array format is a geometrically-regular shape that allows for fabrication, handling, placement, stacking, reagent introduction, detection, and storage. The array can be configured in a row and column format, with regular spacing between each location. Alternatively, the locations can be bundled, mixed, or homogeneously blended for equalized treatment and/or sampling. An array can comprise a plurality of addressable locations configured so that each location is spatially addressable for high-throughput handling, robotic delivery, masking, and/or sampling of reagents and/or by detection means including scanning by laser illumination and confocal and/or deflective light gathering. The array can comprise one or more

"addressable locations," e.g., "addressable positions," that is, physical locations that comprise a known type of molecule.

As used herein, the term "fragment size" refers to the size of a single nucleic acid molecule or of a population of nucleic acid molecules. A population of nucleic acids molecules may comprise a range of sizes having a size distribution. The size distribution may have a "maximum" or "peak" that is the characteristic "fragment size" for the population of nucleic acids.

A "system" denotes a set of components, real or abstract, comprising a whole where each component interacts with or is related to at least one other component within the whole.

Description

Provided herein is technology related to an ultrasonic device comprising temperature control to remove heat from the ultrasonic component and/or to cool a sample subjected to sound waves produced by the ultrasonic device. Although the disclosure herein refers to certain illustrated embodiments, it is to be understood that these embodiments are presented by way of example and not by way of limitation.

For example, in some embodiments the ultrasonic device provides for the fragmentation of nucleic acids (e.g., for preparation of a NGS library) and in some embodiments the ultrasonic device provides for the lysis of cells (e.g., for lysing cells to release nucleic acids). In some embodiments, the ultrasonic device provides a technology related to a step in the preparation of a sample for sequencing. In particular

embodiments, the ultrasonic device provides a fast (e.g., providing fragments in 1 to 5 minutes, e.g., in under 4.5, 4, 3.5, 3, or 2.5 minutes), inexpensive, low-bias method that is easily integrated into a microfluidic cartridge. For embodiments related to cell lysis, ultrasonics provides a method to increase the lysis of difficult samples such as spores, while also improving mixing of samples during and after lysis.

Experiments conducted during the development of the technologies provided herein indicated that the ultrasonic device described reproducibly sheared nucleic acids into fragments of an appropriate size range for the preparation of a NGS library (e.g., approximately 100 to 5000 bp) for input into extant NGS platforms and further indicated that the technology produces longer fragments, e.g., up to tens of kilobase pairs (e.g., up to 10, 20, 30, 40, or 50 kbp) that are appropriate for NGS technologies that process and/or read longer templates and thus provide longer sequence reads.

In some embodiments, the technology comprises an ultrasonic device comprising a chamber designed for cell lysis and/or nucleic acid fragmentation (e.g., comprising a head space and/or a vent (e.g., comprising a HEPA filte) to relieve pressure), a thermoelectric device for active cooling (or, in some embodiments, heating) of the ultrasonic transducer, sensors and hardware/software components to provide data and feedback describing the frequency output of the transducer, and, in some embodiments, grinding beads (e.g., zirconia, glass, ceramic, steel, agate, silica, etc. beads) to aid sample preparation.

Ultrasonic device

The technology provides an ultrasonic device comprising a number of features that are advantageous for processing biological samples. In particular, the ultrasonic device produces sonic energy to process biological samples, e.g., in some embodiments the ultrasonic device produces sonic energy that lyses cells in biological samples, thus releasing nucleic acids (e.g., DNA) for further processing and analysis. In some embodiments, the ultrasonic device produces sonic energy to fragment nucleic acids, e.g., to produce random breaks in an input sample of DNA and provide an output sample comprising DNA fragments having a size distribution that is smaller than the size distribution of the input sample. In some embodiments, the ultrasonic device technology is used for DNA fragmentation as a step in the preparation of a sequencing library as input to a next-generation sequencing workflow. Particular steps comprise lysing cells with sonic energy to release DNA and fragmenting the released DNA with sonic energy.

Particular aspects of the technology include^ l) "dry coupling" of the ultrasonic device to the sample and/or vessel containing the sample (e.g., a microfluidic cartridge), e.g., by providing a tight interaction of the ultrasonic device and the vessel containing the sample (e.g., by a force provided by a spring and/or by a vacuum) and/or by the placement of a hole in the piezoelectric component of the ultrasonic device to provide for a vacuum seal between the piezoelectric component and the vessel holding the sample and or to minimize the air gap between the piezoelectric component and the vessel holding the sample; 2) thermoelectric cooling of the ultrasonic device and/or sample; 3) insulating rings to provide thermal and vibrational isolation from the surroundings (e.g., non-sample and/or non-sample vessel materials, components, modules, and/or devices); and 4) a configuration of the device that provides for the non-focused, random shearing and fragmentation of nucleic acids in the sample.

In some embodiments, the ultrasonic device is placed in close proximity to a sample (e.g., a sample comprising cells and/or nucleic acid) to impart energy (e.g., sonic energy, acoustic energy, etc.) into the sample to lyse the cells and/or fragment the nucleic acids.

In some embodiments, the device comprises a source of sonic (e.g., ultrasonic) energy (e.g., a piezoelectric transducer). In certain embodiments, the sonic energy source (e.g., an ultrasound transducer or other transducer) produces acoustic waves in the

"ultrasonic" frequency range. Ultrasonic waves start at frequencies above those that are audible, typically about 20 kilohertz (kHz) and continue into the frequency region of megahertz (MHz) waves. The speed of sound in water is about 1000 meters per second; thus, the wavelength of a 1000 Hz wave in water is about one meter, which is typically too long for application to small sample sizes. At 20 kHz the wavelength is about 5 cm, which is effective in relatively small treatment vessels. Depending on the sample and vessel volume, preferred frequencies may be higher, for example, about 100 kHz, about 1 MHz, or about 10 MHz, with wavelengths, respectively, of approximately 1.0, 0.1, and 0.01 cm. In contrast, for conventional sonication, including sonic welding, frequencies are typically approximately in the tens of kHz, and for imaging, frequencies are more typically about 1 MHz and up to about 20 MHz. In some embodiments, the frequency used is selected based on the energy absorption characteristics of the sample or of the treatment vessel. To the extent that a particular frequency is better absorbed or preferentially absorbed by the sample, it may be preferred. The energy can be delivered in the form of short pulses or as a continuous field for a defined length of time. The pulses can be bundled or regularly spaced.

In some embodiments, the ultrasonic device comprises a piezoelectric component (e.g., a piezoelectric transducer). A piezoelectric transducer is an electro-mechanical device that interconverts mechanical and electrical energies. The piezoelectric effect is a reversible process - materials exhibiting the direct piezoelectric effect (the internal generation of electrical charge resulting from an applied mechanical force) also exhibit the reverse piezoelectric effect (the internal generation of a mechanical strain resulting from an applied electrical field). As used herein, the term "piezoelectric effect" refers to the generation of mechanical energy (e.g., sound energy) by applying an electric current to a piezoelectric device (e.g., a piezoelectric transducer), which is sometimes known in the art as the "reverse piezoelectric effect". Piezoelectric transducers are available from many commercial producers for off-the-shelf use.

In some embodiments, the piezoelectric transducer is a non-focusing transducer, e.g., for random shearing and fragmenting of nucleic acids in a sample. In general, piezoelectric transducers can be designed to produce either a focused or non-focused beam. A focused beam produces sonic energy pulses having a small diameter and thus high energy concentration, especially in a limited and defined zone referred to as the focal zone. The distance between the transducer and the focal zone is the focal depth. According to certain embodiments of the technology provided, the piezoelectric transducer is an unfocused transducer. An unfoculed transducer produces a beam with two distinct regions^ one is the so-called near field or Fresnel zone and the other is the far field or Fraunhofer zone. In the near field, the sound energy pulse maintains a relatively constant diameter. The length of the near field is related to the diameter D of the transducer and the wavelength L of the ultrasound by:

Near field length = D²/4xL.

One characteristic of the near field is that the intensity along the wave is not constant; in particular, the intensity along the wave oscillates between maximum and zero several times between the transducer and the boundary between the near field and far field due to interference patterns created by the sound waves from the transducer surface. An intensity of zero at a point along the axis indicates that the sound energy is concentrated around the periphery of the beam. A picture of the ultrasound pulse in such a region appears as concentric rings. Further, the beam diverges in the far field, causing the ultrasound pulses to be larger in diameter but to have less intensity along the central axis.

Further, in some embodiments, the piezoelectric transducer comprises a piezoelectric material such as a piezoelectric ceramic. The piezoelectric material is stimulated by application of fluctuating voltages across its thickness to vibrate and so to produce acoustic waves. In some embodiments, the ultrasonic source produces "focused" acoustic waves and in some preferred embodiments the ultrasonic source produces non- focused acoustic waves in the sample. In some embodiments, the sound waves are non- focused into the sample to provide random fragmentation of nucleic acids. Transfer of energy into a sample can be controlled, e.g., by adjusting the parameters of the acoustic wave such as frequency, amplitude, and cycles per burst. In some embodiments, the ultrasonic device comprises an electromagnetic transducer to produce ultrasonic energy. For example, strong pressures of about 16 MPa have been observed in samples exposed to sonic energy produced by an electromagnetic transducer, which provide a source of cavitation bubbles in water and thus are desirable in some embodiments for a lysis and/or fragmentation process.

In some embodiments, the ultrasonic device comprises a capacitive

micromachined ultrasonic transducer (CMUT). See, e.g., Oralkan et al. (2002)

"Capacitive Micromachined Ultrasonic Transducers^ Next- Generation Arrays for Acoustic Imaging?", IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control 49(11): 1596.

In some embodiments, the ultrasonic device comprises a sensor for monitoring the sonic energy or its effect and a feedback mechanism coupled with the source of sonic energy to regulate the energy (for example, voltage, frequency, pattern) for transmitting ultrasonic energy to the sample. Devices for transmission may include detection and feedback circuits to control one or more of losses of energy at boundaries and in transit via reflection, dispersion, diffraction, absorption, dephasing, and detuning. For example, these devices can control energy according to known loss patterns, such as by beam splitting. Sensors can detect the effects of ultrasonic energy on samples, for example, by measuring electromagnetic emissions, typically in the visible, IR, and UV ranges, optionally as a function of wavelength. These effects include energy dispersion, scattering, absorption, and/or fluorescence emission. Other measurable variables include electrostatic properties such as conductivity, impedance, inductance, and/or the magnetic equivalents of these properties. Measurable parameters also include observation of physical uniformity and pattern analysis.

In some embodiments, the ultrasonic device contacts a vessel in which the sample is contained (e.g., a sample holding region or reaction chamber of a microfluidic cartridge, e.g., a cartridge inserted into an apparatus comprising the ultrasonic device). In some embodiments, "dry coupling" is used to couple the ultrasonic device to the sample and/or to the vessel holding the sample (e.g., a microfluidic cartridge). For example, in some embodiments, the ultrasonic device is held or pressed tightly to a vessel holding a sample (e.g., a microfluidic cartridge) by a spring (e.g., a spring-loaded mechanism) or by pressure (e.g., by applying a vacuum) to couple the device to the vessel holding the sample.

During the development of embodiments of the technology provided herein, experiments were conducted to test the fragmentation performance and reduce the performance variability. In particular, the dry coupling of the sample chamber to the piezoelectric transducer was provided by creating a hole in the middle of the

piezoelectric transducer and a vacuum was applied through the hole to bring the sample chamber in contact with the transducer. Accordingly, in some embodiments, the piezoelectric transducer comprises a hole to provide coupling of the transducer with the sample. In some embodiments, the hole minimizes, reduces, or eliminates the production of bubbles in the sample.

Thus, in some embodiments, a gel, paste, or a liquid is not used to couple the ultrasonic device to the vessel holding the sample. Accordingly, in some embodiments the ultrasonic device is "dry coupled" to the sample and/or to the vessel holding the sample (e.g., a microfluidic cartridge). Consequently, the technology provided herein is distinct from existing technologies in which ultrasonic couplants are used to facilitate the transmission of sound energy between the ultrasonic (e.g., piezoelectric) transducer and the sample because sound energy at ultrasonic frequencies is not effectively transmitted through air. For example, a thin air gap between the transducer and the sample typically prevents efficient sound energy transmission and causes severe acoustic impedance in existing technologies. As demonstrated by experiments conducted during the development of embodiments of the technology provided herein, the ultrasonic technology described herein overcomes the problems of air gaps and inefficient transmission of sound energy without the use of a couplant such as a gel, a paste, or a liquid couplant.

Accordingly, in some embodiments, the ultrasonic device comprises a

piezoelectric component comprising a hole (e.g., in its center), e.g. to provide for a vacuum seal that maximizes contact of the piezoelectric component with the vessel holding the sample and/or to minimize the air gap between the piezoelectric component and the vessel holding the sample and/or to minimize, reduce, and/or eliminate the production of bubbles in the sample. During the development of embodiments of the technology provided herein, data were collected demonstrating that the size distribution of fragments changed by introducing a hole in the piezoelectric component. In particular, the distribution of fragment sizes had a peak at approximately 2200 base pairs when nucleic acids were fragmented with a piezoelectric component comprising a hole and a peak at approximately 5000 base pairs when the nucleic acids were fragmented with a piezoelectric component that did not comprises a hole. Further, the piezoelectric component comprising a hole produced fragment libraries having a more reproducible fragment size distribution than a piezoelectric component that did not comprise a hole.

Thermoelectric component

Further, in some embodiments the ultrasonic device comprises a thermoelectric

(e.g., Peltier) component, e.g., to cool the piezoelectric component and/or to cool the sample. As used herein, the term "thermoelectric effect" refers to the direct conversion of temperature differences to electric voltage and vice versa. In particular, a thermoelectric device creates voltage when there is a different temperature on each side of a

semiconductor. Conversely, when a voltage is applied to a thermoelectric device, the device creates a temperature difference (e.g., a movement of thermal energy).

Accordingly, the thermoelectric effect can be used to generate electricity, measure temperature, or change the temperature of objects (e.g., to cool a piezoelectric

transducer and/or a sample). Because the direction of heating and cooling is determined by the polarity of the applied voltage, thermoelectric devices are used as temperature controllers (e.g., to provide thermal energy (e.g., heat) and/or to remove thermal energy (e.g., cool) to another component or system).

While in technical use the term "thermoelectric effect" encompasses three separately identified effects known as the "Seebeck effect", the "Thomson effect", and the "Peltier effect", as used herein the term "thermoelectric effect" refers to the Peltier effect. The Peltier effect refers to the generation or removal of heat at an electrified junction of two different conductors when a current is made to flow through the junction. As used herein, the term "Peltier device" is used interchangeably with "thermoelectric device" to refer to a device that removes heat (e.g., cools) or generates heat as a result of an electric current flowing through the device. In particular embodiments, a

thermoelectric device is used according to the technology to cool a piezoelectric transducer and/or to cool a sample subject to sound energy produced by a piezoelectric transducer.

In particular, during the development of the ultrasonic technology provided herein, it was discovered that the piezoelectric transducers produce heat during the treatment of a sample to lyse cells and/or fragment nucleic acid. In particular, experiments indicated that the heat generated can cause damage to various components with which it is associated in various embodiments, such as a microfluidic cartridge. Heat can also cause leakage of sample and/or denaturation and/or evaporation of the nucleic acid sample. Further, cooling of the piezoelectric element minimizes or eliminates temperature-associated frequency drift in the output of the piezoelectric transducer, thus also improving the reliability and robustness of the ultrasonic technology provided herein. Thus, it was discovered that the ultrasonic device functions better in some applications when it is cooled.

Thermoelectric devices often perform better when a DC voltage is supplied that has a low ripple. Accordingly, in some embodiments, a high speed buffered digital input/output module provides a low ripple DC voltage to the thermoelectric device. Also, in some embodiments the thermoelectric component is mounted on a spring element to maximize its firm contact with a vessel comprising a sample (e.g., a reaction chamber of a microfluidic cartridge). In some embodiments the thermoelectric component is held in firm contact with a vessel comprising a sample (e.g., a reaction chamber of a microfluidic cartridge) by gas pressure (e.g., by application of a vacuum, e.g., through a latex ring seal between the thermoelectric device and the vessel holding a sample). In some embodiments, the thermoelectric device comprises a coating (e.g., a polymer coating such as, e.g., a PTFE coating, a perfluoroalkoxy (PFA) coating, a fluorinated ethylene propylene (FEP) coating, a polychlorotrifluoroethylene coating, a tetrafluorethylene- perfluoropropylene coating, a polyether ether ketone (PEEK) coating, or a nylon coating), to minimize or eliminate sticking of the thermoelectric device to a vessel comprising a sample. In some embodiments, an insulating ring surrounds the

thermoelectric device to reduce heat leakage and/or to increase the speed of temperature changes. In some embodiments, the insulating ring surrounding the thermoelectric device is the same insulating ring that surrounds the source of ultrasonic energy to provide isolation from vibration and sound.

Particular embodiments provide an ultrasonic device comprising a thermoelectric component that removes heat from the sample to maintain the sample at less than approximately 99°C, 90°C, 85°C, 80°C, 85°C, 80°C, or 75°C (e.g., less than 74.9, 74.8, 74.7, 74.6, 74.5, 74.4, 74.3, 74.2, 74.1, 74, 73, 72, 71, 70, 65, 60, or less than 60°C). In some embodiments, the thermoelectric component removes heat from the sample to minimize or eliminate denaturation of nucleic acid (e.g., DNA) during fragmentation of the nucleic acid (e.g., DNA).

In addition, some embodiments comprise a piezoelectric transducer that functions at a frequency of 1 to 1000 kHz. In some embodiments, the piezoelectric transducer functions at a frequency of from 1 to 1000 MHz. In some embodiments, the ultrasonic transducer is a transducer that produces frequencies in the MHz range (e.g., 1 to 1000 MHz, e.g., 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 MHz, e.g., in some embodiments from 1 to 10, e.g., 1 to 5, e.g., 1 to 2, e.g., 1.7 MHz).

In some embodiments, the ultrasonic device comprises a resistive heater, e.g., to increase the speed of heat transfer. In some embodiments, multiple thermoelectric devices are linked in series (e.g., one thermoelectric device heats and/or cools the heat sink of another thermoelectric device). In some embodiments, the thermoelectric device is a high power (e.g., approximately 8 to 12 W, e.g., 8, 9, 10, 11, or 12 W, e.g., a 9 W) thermoelectric device that heats at approximately twice the speed of a lower power (e.g., a 4.5-W) thermoelectric device. In some embodiments, thermoelectric heating elements are positioned on multiple sides of a sample chamber (e.g., a reaction chamber) to "sandwich" the reaction chamber.

Insulating ring

In some embodiments, the ultrasonic device comprises an insulating ring to provide thermal and vibrational isolation from the surroundings (e.g., non-sample and or non-sample vessel materials, components, modules, and/or devices). In some embodiments, the insulating ring comprises silicone to seal the ultrasonic device to a vessel holding a sample. In some embodiments, the insulating ring is made of a heat- resistant and or a fire-resistant material.

In some embodiments the insulating ring comprises a resin-reinforced, glass- reinforced, or polymer-reinforced laminate material, e.g., material comprising a woven fiber component and a resin component. In some embodiments, the insulating ring comprises a material that is a glass-reinforced epoxy. For instance, in some

embodiments, the material is a flame resistant (self-extinguishing) material designated as "FR-4". "FR" stands for "flame retardant" and denotes that the material complies with the standard UL94V-0 (National Electrical Manufacturers Association, 1968). An exemplary FR-4 material is a high-pressure thermoset, plastic laminate composite material comprising woven fiberglass cloth and an epoxy resin binder. Exemplary embodiment of an ultrasonic device

An exemplary embodiment of an ultrasonic device 100 (e.g., as shown in Figure l) comprises a piezoelectric component (e.g., a 1.7 MHz piezoelectric component) 110 attached to a thermoeletric component 120. In some embodiments, the ultrasonic device comprises one or more temperature sensors 130 and appropriate wiring to provide current to the sensors and to carry a signal (e.g., a voltage or current) from the sensors, e.g., to an analog-to- digital converter and/or to a computer. In some embodiments, the ultrasonic device comprises a seal (e.g., a silicone ring) and/or an insulating ring 140 (e.g., comprising silicone and/or a (e.g., heat resistant) composite material comprising fiberglass and epoxy (e.g., an FR4 flame retardant material)) for flexible mounting, sealing, and isolation/insulation of temperature and vibration from other components and modules. In some embodiments, the ultrasonic device further comprises a thermal interface 150 for thermal communication to a heat sink to remove thermal energy from the thermoelectric device efficiently. The device comprises appropriate wiring or cables 160 to provide power to the piezoelectric component 110, thermoelectric component 120, temperature sensors 130, etc. and to transmit signals from the temperature sensors, e.g., to a microcontroller or microprocessor. In some embodiments the device comprises an analog-to-digital (A/D) converter, e.g., to convert an analog voltage output from a temperature sensor 130 to a digital (e.g., voltage) signal.

In some embodiments, the device comprises a power supply to provide a voltage and current to the device components. In some embodiments, the ultrasonic device comprises a frequency regulator. In some embodiments, the device comprises a relay to switch the direction of current provided to the thermoelectric device 120 (e.g., to switch the device from providing thermal energy (e.g., heating) to removing thermal energy (e.g., cooling) and to switch the device from removing thermal energy (e.g., cooling) to providing thermal energy (e.g., heating)). In some embodiments, the device comprises a high-speed buffered digital input/output to provide a low ripple DC voltage to the thermoelectric component 120. In some embodiments, the ultrasonic device comprises a spring mounting or vacuum line to maintain a tight seal and connection of the ultrasonic device to a vessel comprising a sample (e.g., a microfluidic cartridge). In some embodiments, the thermoelectric component of the ultrasonic device comprises a coating (e.g., a polymer coating such as, e.g., a PTFE coating, a perfluoroalkoxy (PFA) coating, a fluorinated ethylene propylene (FEP) coating, a polychlorotrifluoroethylene coating, a tetrafluorethylene-perfluoropropylene coating, a polyether ether ketone (PEEK) coating, or a nylon coating), e.g., to reduce adherence (e.g., "sticking") of the thermoelectric device to a vessel comprising a sample (e.g., a microfluidic cartridge).

Sample chamber

In some embodiments the sample is present in a cartridge and the ultrasonic device is used to process the sample in the cartridge. In some embodiments, the sample is contained in a sample chamber comprising a vent to release pressure produced by ultrasonic treatment of the sample. In some embodiments, the sample chamber comprises a vent comprising a filter (e.g., a HEPA filter) to prevent sample components from being released from the sample chamber.

For example, experiments conducted during the development of embodiments of the technology provided herein indicated that pressure increased in the vessels containing the samples (e.g., a microfluidic cartridge, e.g., a reaction chamber in a microfluidic cartridge) to a level that damaged the vessels. In particular, experiments indicated that high pressures caused microfluidic vessels to break and/or to delaminate.

The technology is provided as a device to lyse cells and/or fragment nucleic acids. Accordingly, the technology provides sonic energy into a sample chamber comprising cells that are to be lysed and/or into a sample chamber comprising nucleic acid molecules that are to be fragmented. In some embodiments, cell lysis and nucleic acid fragmentation occur in the same sample chamber and in some embodiments cell lysis and nucleic acid fragmentation occur in separate sample chambers.

As such, in some embodiments the sample chamber (e.g., a nucleic acid fragmentation chamber) has a volume of approximately 1000 to 2000 mm³ (e.g., 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mm³, preferably approximately 1500 mm³). In some embodiments the sample chamber (e.g., a cell lysis chamber) has a volume of approximately 6200 mm³ (e.g., 5000 to 7500 mm³, e.g., 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, or 7500 mm³). 1 mm³ is equivalent to 0.001 milliliter (l microliter) and thus volumes in mm³ and milliliters, microliters, picoliters, nanoliters, etc. are readily interconvertible.

In some embodiments the sample volume to the sample chamber volume is approximately 1=5 to 1=20 (e.g., 1=5, 1=6, 1=7, 1=8, 1=9, 1 = 10, 1=11, 1=12, 1=13, 1=14, 1 = 15, 1:16, 1:17, 1:18, 1^19, or 1:20, preferably approximately 1:12).

In some embodiments, the top of the chamber comprises a vented channel connected to an adjacent chamber for pressure release and to collect aerosols and thus contain the sample from release outside the sample chamber. In some embodiments, the vented channel comprises a filter (e.g., an aerosol barrier filter, e.g., a HEPA filter).

In some embodiments, the sample chamber comprises a particle or bead to increase cavitation and/or mixing of a sample (e.g., during lysis of cells, during fragmentation of nucleic acids, etc.) In some embodiments, the particle or bead is a grinding bead such as, e.g., hardened steel beads, stainless steel beads, tungsten carbide beads, agate beads, zirconium oxide beads, PTFE beads, yttrium stabilized zircon beads, glass beads, or silica beads, or a mixture of particles or beads. In some embodiments, the particle or bead is a ceramic particle or bead. In some embodiments, the particle or bead is a yttrium stabilized zirconia (ZY) bead. In some embodiments, the beads have a diameter of approximately 2 mm (e.g., 1 to 5 mm).

In some embodiments, the sample chamber comprises a chaotropic salt and/or a proteinase (e.g., proteinase K), e.g., to enhance the cell lysis process. The combination of chemical and physical lysis processes provides for the efficient lysis of a wide range of cell types. Sample

The technology relates to the processing of samples, e.g., biological samples. Thus, the technology is related to the interaction of the ultrasonic device with a sample and/or the delivery of a sample to the ultrasonic device, e.g., in a vessel containing the sample (e.g., a microfluidic cartridge comprising the sample, e.g., a reaction chamber (e.g., a cell lysis chamber and/or a nucleic acid fragmentation chamber) in a microfluidic cartridge).

Examples of samples include various fluid samples. In some instances, the sample is a bodily fluid sample from the subject. In some embodiments, the sample is an aqueous or a gaseous sample. In some embodiments, the sample is a gel. In some embodiments, the sample includes one or more fluid component. In some embodiments, solid or semi- solid samples are provided. In some embodiments, the sample comprises tissue collected from the subject. In some embodiments, the sample comprises a bodily fluid, secretion, and/or tissue of a subject. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is a bodily fluid, a secretion, and/or a tissue sample. Examples of biological samples include but are not limited to, blood, serum, saliva, urine, gastric and digestive fluid, tears, stool, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, sweat, mucus, earwax, oil, glandular secretions, breath, spinal fluid, hair, fingernails, skin cells, plasma, nasal swab or nasopharyngeal wash, spinal fluid, cerebral spinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, cord blood, emphatic fluids, cavity fluids, sputum, pus, micropiota, meconium, breast milk, and/or other excretions. In some embodiments, the sample is provided from a human or an animal, e.g., in some embodiments the sample is provided from a mammal (e.g., a vertebrate) such as a murine, simian, human, farm animal, sport animal, or pet. In some embodiments, the sample is collected from a living subject and in some embodiments the sample is collected from a dead subject.

In some embodiments, the sample is collected fresh from a subject and in some embodiments the sample has undergone some form of pre-processing, storage, or transport. For example, in some embodiments the sample is a formalin or formaldehyde fixed paraffin embedded (FFPE) sample. FFPE samples (e.g., FFPE tissue samples) provide for the stable storage of nucleic acids. The clinical utility of FFPE samples is substantial, where retrospective analysis of archival tissue enables the correlation of molecular findings with the response to treatment and the clinical outcome.

In some embodiments, the sample comprises nucleic acids that are amplified, e.g., prior to or after a fragmentation step. In some embodiments, the sample is provided to an ultrasonic device from a subject without undergoing intervention or much time. In some embodiments, the subject contacts the ultrasonic device, a removable cartridge, and/or a vessel to provide the sample.

In some embodiments, a subject provides a sample and/or the sample may be collected from a subject. In some embodiments, the subject is a patient, clinical subject, or pre-clinical subject. In some embodiments, the subject is undergoing diagnosis, treatment, and/or disease management or lifestyle or preventative care. The subject may or may not be under the care of a health care professional.

In some embodiments, the sample is collected from the subject by puncturing the skin of the subject or without puncturing the skin of the subject. In some embodiments, the sample is collected through an orifice of the subject. In some embodiments, a tissue sample (e.g., an internal or an external tissue sample) is collected from the subject. In some embodiments, the sample is collected from a portion of the subject including, but not limited to, the subject's finger, hand, arm, shoulder, torso, abdomen, leg, foot, neck, ear, or head.

In some embodiments, the sample is an environmental sample. Examples of environmental samples include, but are not limited to, air samples, water samples, soil samples, or plant samples. Additional samples include food products, beverages, manufacturing materials, textiles, chemicals, therapies, or any other samples.

In some embodiments, the sample is mixed with a chaotropic salt and/or a proteinase (e.g., proteinase K), e.g., to enhance the cell lysis process.

In some embodiments, one type of sample is accepted and/or processed by the ultrasonic device. Alternatively, in some embodiments multiple types of samples are accepted and/or processed by the ultrasonic device. For example, in some embodiments the ultrasonic device is capable of accepting one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twelve or more, fifteen or more, twenty or more, thirty or more, fifty or more, or one hundred or more types of samples. In some embodiments, the ultrasonic device is capable of accepting and/or processing any of these numbers of sample types

simultaneously and/or at different times from different or the same matrices. For example, in some embodiments the ultrasonic device is capable of preparing, assaying and or detecting one or multiple types of samples.

The technology is not limited in the volume of sample that is processed by the ultrasonic device. Accordingly, embodiments provide that any volume of sample is provided from the subject or from another source. Examples of volumes may include, but are not limited to, approximately 10 mL or less, 5 mL or less, 3 mL or less, 1 mL or less, 500 μϊ-ι or less, 300 μΐι or less, 250 μΐι or less, 200 μΐι or less, 170 μΐ^ or less, 150 μΐ^ or less, 125 μΐι or less, 100 μΐι or less, 75 μΐι or less, 50 μΐ^ or less, 25 μΐι or less, 20 μΐ^ or less, 15 μΐ^ or less, 10 μΐι or less, 5 μΐι or less, 3 μΐ^ or less, 1 μΐι or less, 500 nL or less, 250 nL or less, 100 nL or less, 50 nL or less, 20 nL or less, 10 nL or less, 5 nL or less, 1 nL or less, 500 pL or less, 100 pL or less, 50 pL or less, or 1 pL or less. The amount of sample may be approximately a drop of a sample. The amount of sample may be approximately 1 to 5 drops of sample, 1 to 3 drops of sample, 1 to 2 drops of sample, or less than a drop of sample. The amount of sample may be the amount collected from a pricked finger or fingerstick. Any volume, including those described herein, is provided to the device in various embodiments.

Further, in some embodiments a sample collection unit and/or sample reaction chamber is integral to the ultrasonic device. And, in some embodiments the sample collection unit and/or sample reaction chamber is separate from the ultrasonic device. In some embodiments, the sample collection unit and/or sample reaction chamber is removable and/or insertable from the ultrasonic device or is removable and/or insertable from an apparatus comprising the ultrasonic device. In some embodiments, the sample collection unit and/or sample reaction chamber is provided in a cartridge; in some embodiments the sample collection unit and/or sample reaction chamber is not provided in a cartridge. In some embodiments, a cartridge is removable and/or insertable from the ultrasonic device or is removable and/or insertable from an apparatus comprising the ultrasonic device. In some embodiments, a cartridge is not removable and/or not insertable from the ultrasonic device or is not removable and/or not insertable from an apparatus comprising the ultrasonic device. In some embodiments a sample collection unit and/or sample reaction chamber is configured to receive a sample. In some embodiments, the sample collection unit is capable of containing and/or confining the sample. In some embodiments, the sample collection unit is capable of conveying the sample to the ultrasonic device and/or sample chamber associated (e.g., coupled to) the ultrasonic device.

In some embodiments, an ultrasonic device is configured to accept a single sample; in some embodiments an ultrasonic device is configured to accept multiple samples. In some embodiments, the multiple samples comprise multiple types of samples. For example, in some embodiments a single ultrasonic device handles a single sample at a time. For example, in some embodiments an ultrasonic device receives a single sample and performs one or more sample processing steps, such as a lysis steps, isolation step, and/or a fragmentation step with the sample. In some embodiments, the ultrasonic device completes processing a sample before accepting a new sample.

In other embodiments, an ultrasonic device is capable of handling multiple samples simultaneously. In one example, the ultrasonic device receives multiple samples simultaneously. In some embodiments, the multiple samples comprise multiple types of samples. Alternatively, in some embodiments the ultrasonic device receives samples in sequence. Samples are provided in some embodiments to the ultrasonic device one after another or, in some embodiments, samples are provided to the ultrasonic device after any amount of time has passed. An ultrasonic device in some embodiments begins sample processing on a first sample, receives a second sample during said sample processing, and processes the second sample in parallel with the first sample. In some embodiments, the first and second samples are not the same type of sample. In some embodiments, the ultrasonic device processes any number of samples in parallel, including but not limited to more than and/or equal to approximately one sample, two samples, three samples, four samples, five samples, six samples, seven samples, eight samples, nine samples, ten samples, eleven samples, twelve samples, thirteen samples, fourteen samples, fifteen samples, sixteen samples, seventeen samples, eighteen samples, nineteen samples, twenty samples, twenty-five samples, thirty samples, forty samples, fifty samples, seventy samples, one hundred samples.

In some embodiments, the ultrasonic device comprises one, two, or more modules that are process one, two, or more samples in parallel. The number of samples that are processed in parallel may be determined by the number of available modules and/or components in the device. When a plurality of samples is processed simultaneously, embodiments provide that the samples begin and/or end processing at any time. For example, the samples need not begin and/or end processing at the same time. In some embodiments, a first sample has completed processing while a second sample is still being processed. In some embodiments, the second sample has begun processing after the first sample has begun processing. As samples have completed processing, additional samples are added to the device in some embodiments. In some embodiments, the device runs continuously with samples being added to the device as various samples have completed processing.

In some embodiments, multiple samples are provided simultaneously. In some embodiments, multiple samples are not the same type of sample. In some embodiments, multiple sample collection units are provided to an ultrasonic device. In some

embodiments, the multiple sample collection units receive samples simultaneously and in some embodiments the multiple sample collection units receive samples at different times. In some embodiments, multiples of any of the sample collection mechanisms described herein are used in combination.

In some embodiments, multiple samples are provided in sequence. In some embodiments, multiple sample collection units are used and in some embodiments single sample collection units are used. Embodiments provide any combination of sample collection mechanisms described herein. In some embodiments, an ultrasonic device accepts one sample at a time, two samples at a time, or more. In some

embodiments, samples are provided to the ultrasonic device after any amount of time has elapsed.

Cell lysis

Acoustic energy can disrupt cells. It is generally thought there are two ways in which ultrasound affects cells: heating and cavitation (e.g., the interaction of a sound wave with small gas bubbles in the sample). Heating occurs primarily due to absorption of the sound energy by the medium or by the container. For dilute aqueous systems, it is absorption by the container that is a main source of the heating. Heating is not desirable in some treatment applications, as described herein.

According to the technology, ultrasonic waves are used to treat a sample containing biological material. The ultrasonic waves can be specifically adapted to interact preferentially with supporting matrices in a biological material, such as plant cell walls or extracellular matrices such as bone or collagen, thereby lessening or removing a barrier function of such matrices and, in some embodiments, facilitating lysis. Other modes of sonic energy can have different effects than disrupting a matrix and can be used either with pre-treatment, with disrupting sonic energy, or by themselves. For, example the conditions to disrupt a matrix can be different from those to permeabilize a cell membrane.

There are many possible mechanisms by which cavitation may affect cells and there is no consensus as to which mechanisms, if any, dominate. The principle mechanisms are thought to include shear, microjets, shock waves, sonochemistry, and other mechanisms, as discussed more fully below.

Shear: Significant shear forces are associated with the violent collapse of bubbles. Because cell membranes are sensitive to shear, it is thought that cavitation may permeabilize cell membranes. In some cases, the membrane is apparently permeable for only a short time, during which molecules may be passed into or out of the cell. In other cases the cell is lysed.

Microjets^ Bubbles undergoing a violent collapse, particularly near a boundary, such as a container wall, typically collapse asymmetrically and generate a liquid jet of fluid that passes through the bubble and into the boundary. The speed of this jet has been measured to be hundreds of meters a second and is of great destructive power. It may play a major role in the destruction of kidney stones by acoustic shock waves and may be a possible way of destroying blood clots.

Shock Wave: Collapse of a bubble spherically can generate an intense shock wave. This pressure can be thousands of atmospheres in the neighborhood of the bubble. The compressive stress of the shock wave may be strong enough to cause cellular material to fail.

Sonochemistry: The pressure and temperatures in the bubble during an inertial collapse can be extraordinarily high. In extreme examples, the gas can be excited sufficiently to produce light, termed sonoluminescence. Although the volume is small and the time duration short, this phenomenon has been exploited to enhance chemical reaction rates. The production of free-radicals and other sonochemicals may also affect cells.

Other: Other factors also may be involved. Vessel walls may contribute cavitation nuclei. A plastic vessel with an aqueous fluid may result in a standing wave field due to internal reflections, as a result of impedance mismatches between the fluid and the vessel walls. In some embodiments cell contents are stirred to increase cell lysis. Mixing moves bubbles from the edges of a reaction chamber to be brought into contact with a cell or a tissue. This mixing promotes inertial, transient acoustic cavitation near the cell walls, resulting in cellular lysis.

Nucleic acid fragmentation

Nucleic acid shearing or fragmentation is a step in several embodiments for constructing nucleic acid libraries and in embodiments for other molecular biological technologies (e.g., hybridization of target nucleic acids on solid supports, for example, microarrays) . Accordingly, embodiments are provided for controlled shearing and fragmentation of nucleic acids, e.g., to provide increased efficiency in preparation of nucleic acids for subsequent analysis. In particular, fragmenting nucleic acids (e.g., DNA or RNA) is typically a step of technologies for preparing nucleic acids for next- generation sequencing platforms such as those by Solexa (Illumina), Pacific Biosciences, and Ion Torrent (Life Technologies). The technology provided herein is related to the production of sequencing libraries for any of these or other platforms (either extant or yet to be developed). The technology provided herein can be adapted to provide appropriate efficiency of fragmentation, fragmentation time, fragment length

distribution range, and quality of fragments generated.

Thus, provided herein is technology related to preparing NGS sequencing libraries by fragmenting nucleic acids (e.g., DNA and RNA) using sonic (e.g., acoustic) energy. The fragmenting of a nucleic acid molecule is achieved through the

hydrodynamic action of the liquid on the nucleic acid molecule. When a velocity gradient exists within a liquid medium, the shear stresses produced by the elongational components of flow produce an aligning and extensional action on the nucleic acid molecules along the direction of the shear stresses. When the applied hydrodynamic action (tensile forces, bending moments, etc.) exceeds the intrinsic strength of the polymeric chain, a breakage in the chain will result, giving rise to two fragments, each shorter than the original.

The technology is related to producing varying lengths of fragments, e.g., to provide as input to various sequencing platforms. For example, a paired-end approach typically requires 200 to 500 bp fragments and a mate-pair approach typically requires 2 to 5 kbp fragments. As additional examples, the Solexa 1G sequencing platform requires fragments of approximately 100 to 300 bp and SOLiD requires fragments for library preparation is size ranges of 60 to 90 bp. Sonication parameters (such as power, fragmentation time, duty cycle, cycles per burst, etc.) can be adjusted to adjust the size of fragments produced. The sonication region shape and volume can be modified with changes in the design and fabrication of the vessel containing the sample and the coupling of the ultrasonic transducer to the sample and vessel.

In particular, fragmentation time can be adjusted to increase or decrease the total amount of energy that is provided to the sample and thus modulate the fragment size and/or fragment size distribution. As used herein, "fragmentation time" refers to the length of time sonic energy is provided to a sample. In particular embodiments, fragmentation time ranges from 1 minute to 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 minutes. Experiments were conducted during the development of certain embodiments of the technology provided and data were collected indicating that a fragmentation time of 10 minutes produces fragments of approximately 2000 to 3000 bp using a current of 0.3 amperes. Further, the data indicated that additional time did not produce smaller fragments.

Further, the current supplied to the ultrasonic component (e.g., to the

piezoelectric component) can be adjusted to increase or decrease the sonic energy provided to the sample and thus modulate the fragment size and/or fragment size distribution. In particular embodiments, the current ranges from 0.1 to 1 ampere, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 ampere. Experiments were conducted during the development of certain embodiments of the technology provided and data were collected indicating that a current of 0.3 amperes produces fragments of approximately 2000 to 3000 bp without introducing excessive heat into the sample that may cause the sample to degrade (e.g., without heating the sample to greater than approximately 70°C). For example, the data indicated that a current of 0.35 amperes or 0.4 amperes heated the sample to approximately 60°C to 65°C after 10 minutes of fragmentation time. As such, preferred embodiments use a current that does not reach a temperature of approximately 70°C in a fragmentation time of 10 minutes, e.g., a current of approximately 0.3 amperes.

In addition, the duty cycle may be changed to increase or decrease the sonic energy provided to the sample and thus modulate the fragment size and/or fragment size distribution. As used herein, the term "duty cycle" refers to the percentage of one period in which a signal is active. A period is the time it takes for a signal to complete an on-and-off cycle. For instance, a 60% duty cycle means the signal is on 60% of the time but off 40% of the time. The "on time" for a 60% duty cycle could be a fraction of a second, a day, or even a week, depending on the length of the period. In some

embodiments of the technology provided herein, the duty cycle (e.g., the "on time") is a function of the temperature of the sample and/or piezoelectric component. In particular, in some embodiments a maximum set temperature is used so that when a sensor detects a temperature in the sample and/or in the piezoelectric component that is the maximum set temperature, power (e.g., current) to the piezoelectric component is stopped, thus stopping the delivery of sonic energy to the sample. When the sensor detects a temperature in the sample and/or in the piezoelectric component that is a minimum set temperature, power (e.g., current) to the piezoelectric component is started, thus providing the delivery of sonic energy to the sample. Thus, a lower maximum set temperature results in the piezoelectric component being in a "turned off state more often that a relatively higher maximum set temperature that allows the piezoelectric component to be active for more time. Experiments were conducted during the development of certain embodiments of the technology provided and data were collected indicating that reducing the maximum set temperature, e.g., to below 60°C (e.g., 55°C, 50°C, 45°C, 40°C, or below) resulted in fragments having a size of greater than 4000 bp and resulted in less reproducibility among independent experiments to test

fragmentation. Thus, the maximum temperature set point for the sample in preferred embodiments is approximately 60°C to 70°C.

Accordingly, embodiments provide for the production of nucleic acid fragments over a range of sizes. Particular embodiments provide an ultrasonic device that generates nucleic acid fragments having a size that is approximately 100 bp to 5000 bp (e.g., 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, 550 bp, 600 bp, 650 bp, 700 bp, 750 bp, 800 bp, 850 bp, 900 bp, 950 bp, 1000 bp, 1050 bp, 1100 bp, 1150 bp, 1200 bp, 1250 bp, 1300 bp, 1350 bp, 1400 bp, 1450 bp, 1500 bp, 1550 bp, 1600 bp, 1650 bp, 1700 bp, 1750 bp, 1800 bp, 1850 bp, 1900 bp, 1950 bp, 2000 bp, 2050 bp, 2100 bp, 2150 bp, 2200 bp, 2250 bp, 2300 bp, 2350 bp, 2400 bp, 2450 bp, 2500 bp, 2550 bp, 2600 bp, 2650 bp, 2700 bp, 2750 bp, 2800 bp, 2850 bp, 2900 bp, 2950 bp, 3000 bp, 3050 bp, 3100 bp, 3150 bp, 3200 bp, 3250 bp, 3300 bp, 3350 bp, 3400 bp, 3450 bp, 3500 bp, 3550 bp, 3600 bp, 3650 bp, 3700 bp, 3750 bp, 3800 bp, 3850 bp, 3900 bp, 3950 bp, 4000 bp, 4050 bp, 4100 bp, 4150 bp, 4200 bp, 4250 bp, 4300 bp, 4350 bp, 4400 bp, 4450 bp, 4500 bp, 4550 bp, 4600 bp, 4650 bp, 4700 bp, 4750 bp, 4800 bp, 4850 bp, 4900 bp, 4950 bp, or 5000 bp).

In some embodiments, the technology (e.g., devices, methods, systems, etc.) provides nucleic acid fragments (e.g., for NGS technologies) that are in the ranges of 1 kbp to 5 kbp, 10 kbp, 15 kbp, 20 kbp, 30 kbp, 40 kbp, and/or 50 kbp (e.g., approximately 1000 bp; 2000 bp; 3000 bp; 4000 bp; 5000 bp; 6000 bp; 7000 bp; 8000 bp; 9000 bp; 10,000 bp; 11,000 bp; 12,000 bp; 13,000 bp; 14,000 bp; 15,000 bp; 16,000 bp; 17,000 bp; 18,000 bp; 19,000 bp; 20,000 bp; 25,000 bp; 30,000 bp; 35,000 bp; 40,000 bp; 45,000 bp; or 50,000 bp).

Systems

Some embodiments of the technology are related to systems for processing a biological sample (e.g., a sample comprising cells and/or nucleic acid). Particular embodiments provide systems for lysing cells of a biological sample and fragmenting nucleic acids as a step in the production of a sequencing library for input into an NGS workflow. Embodiments of an ultrasonic device described herein find use in the lysing and fragmenting steps. Accordingly, embodiments of the technology provide a system for production of a NGS library, the system comprising an ultrasonic device to lyse cells and or to fragment nucleic acids and a vessel containing a sample. In some embodiments the system comprises an ultrasonic device to lyse cells and a second ultrasonic device to fragment nucleic acids. In some embodiments the system comprises an ultrasonic device to lyse cells and to fragment nucleic acids. In some embodiments a system comprises a piezoelectric component to generate sound energy and a thermoelectric component to cool the piezoelectric component and/or to cool the sample. In some embodiments a system comprises a piezoelectric component to generate sound energy and a

thermoelectric component to cool the sample.

Some embodiments of systems for the production of a NGS library from a biological sample comprise a sample chamber to hold a biological sample, a piezoelectric component to provide sound energy into the sample (e.g., to lyse cells and/or to fragment nucleic acids), and a thermoelectric component to cool the sample. In some embodiments the system generates nucleic acid fragments having a size that is approximately 100 bp to 5000 bp (e.g., 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, 550 bp, 600 bp, 650 bp, 700 bp, 750 bp, 800 bp, 850 bp, 900 bp, 950 bp, 1000 bp, 1050 bp, 1100 bp, 1150 bp, 1200 bp, 1250 bp, 1300 bp, 1350 bp, 1400 bp, 1450 bp, 1500 bp, 1550 bp, 1600 bp, 1650 bp, 1700 bp, 1750 bp, 1800 bp, 1850 bp, 1900 bp, 1950 bp, 2000 bp, 2050 bp, 2100 bp, 2150 bp, 2200 bp, 2250 bp, 2300 bp, 2350 bp, 2400 bp, 2450 bp, 2500 bp, 2550 bp, 2600 bp, 2650 bp, 2700 bp, 2750 bp, 2800 bp, 2850 bp, 2900 bp, 2950 bp, 3000 bp, 3050 bp, 3100 bp, 3150 bp, 3200 bp, 3250 bp, 3300 bp, 3350 bp, 3400 bp, 3450 bp, 3500 bp, 3550 bp, 3600 bp, 3650 bp, 3700 bp, 3750 bp, 3800 bp, 3850 bp, 3900 bp, 3950 bp, 4000 bp, 4050 bp, 4100 bp, 4150 bp, 4200 bp, 4250 bp, 4300 bp, 4350 bp, 4400 bp, 4450 bp, 4500 bp, 4550 bp, 4600 bp, 4650 bp, 4700 bp, 4750 bp, 4800 bp, 4850 bp, 4900 bp, 4950 bp, or 5000 bp).

In some embodiments the thermoelectric component maintains the sample at a temperature that is less that 99°C, 95°C, 90°C, 85°C, 80°C, or 75°C (e.g., less than 74.9, 74.8, 74.7, 74.6, 74.5, 74.4, 74.3, 74.2, 74.1, 74, 73, 72, 71, 70, 65, 60, or less than 60°C).

In some embodiments of systems a microfluidic cartridge comprises the sample chamber. Accordingly, some embodiments of systems for the production of a NGS library from a biological sample comprise a microfluidic cartridge (e.g., comprising a sample chamber to hold a biological sample), a piezoelectric component to provide sound energy into the sample (e.g., to lyse cells and/or to fragment nucleic acids), and a thermoelectric component to cool the sample. Some embodiments provide an apparatus that comprises the ultrasonic device and that accepts a microfluidic cartridge. Thus, some embodiments of systems comprise an apparatus comprising an ultrasonic device to process a biological sample and a microfluidic cartridge comprising a sample chamber to contain the biological sample. Particular embodiments comprise^ l) an apparatus comprising an interface to accept a microfluidic cartridge comprising a sample chamber, a piezoelectric component, and a thermoelectric component; and 2) a microfluidic cartridge comprising a sample chamber.

In some embodiments, the system comprises a power supply to provide a voltage and current to the system components. In some embodiments, the system comprises a frequency regulator. In some embodiments, the system comprises a relay to switch the direction of current provided to the thermoelectric device (e.g., to switch the device from providing thermal energy (e.g., heating) to removing thermal energy (e.g., cooling) and to switch the device from removing thermal energy (e.g., cooling) to providing thermal energy (e.g., heating)). In some embodiments, the system comprises a high-speed buffered digital input/output to provide a low ripple DC voltage to the thermoelectric component. In some embodiments, the system comprises a spring mounting or vacuum line to maintain a tight seal and connection of the ultrasonic device to a vessel comprising a sample (e.g., a microfluidic cartridge). In some embodiments, the thermoelectric component of the ultrasonic device comprises a coating (e.g., a polymer coating such as, e.g., a PTFE coating, a perfluoroalkoxy (PFA) coating, a fluorinated ethylene propylene (FEP) coating, a polychlorotrifluoroethylene coating, a

tetrafluorethylene-perfluoropropylene coating, a polyether ether ketone (PEEK) coating, or a nylon coating), e.g., to reduce adherence (e.g., "sticking") of the thermoelectric device to a vessel comprising a sample (e.g., a microfluidic cartridge).

Some embodiments provide systems for sequencing a nucleic acid (e.g., using NGS). For example, some embodiments provide a system comprising an apparatus for producing an NGS library for input to an NGS workflow and an NGS sequencer. In some embodiments the NGS sequencer and apparatus are fluidly connected, e.g., by a tube or other conduit for transporting the output NGS library from the apparatus to the input of the NGS sequencer. Particular embodiments of systems for sequencing a nucleic acid comprise^ l) an apparatus comprising an interface to accept a microfluidic cartridge comprising a sample chamber, a piezoelectric component, and a thermoelectric component; 2) a microfluidic cartridge comprising a sample chamber; and 3) an NGS sequencer.

In some embodiments, the methods and systems described herein are associated with a programmable machine designed to receive input from one or more sensors, receive input from a user, provide a voltage or current to an ultrasonic device or a component of an ultrasonic device, and/or perform a sequence of arithmetic or logical operations as provided by the methods described herein.

For example, some embodiments of the technology are associated with (e.g., implemented in) computer software and/or computer hardware. In one aspect, the technology relates to a computer comprising a form of memory, an element for performing arithmetic and logical operations, and a processing element (e.g., a processor or a microprocessor) for executing a series of instructions (e.g., a method as provided herein) to read, manipulate, and store data. Some embodiments comprise one or more processors. In some embodiments, a processor provides instructions to one or more ultrasonic device(s), one or more module(s) of an ultrasonic device, one or more component(s) of an ultrasonic device (e.g., to the piezoelectric component and/or to the thermoelectric component), and/or one or more portion(s) of a component of an ultrasonic device. In some embodiments, a processor receives signals that are detected from one or more sensors (e.g., a temperature sensor, a fluorescence sensor, a vibration sensor, etc.). In some embodiments, a microprocessor is part of a system comprising one or more of a CPU, a graphics card, a user interface (e.g., comprising an output device such as a display and an input device such as a keyboard), a storage medium, and memory components. Memory components (e.g., volatile and/or nonvolatile memory) find use in storing instructions (e.g., an embodiment of a process as provided herein) and/or data. Programmable machines associated with the technology comprise conventional extant technologies and technologies in development or yet to be developed (e.g., a quantum computer, a chemical computer, a DNA computer, an optical computer, a spintronics based computer, etc.).

Some embodiments provide a computer that includes a computer-readable medium. The embodiment includes a random access memory (RAM) coupled to a processor. The processor executes computer-executable program instructions stored in memory. Such processors may include a microprocessor, an ASIC, a state machine, or other processor, and can be any of a number of computer processors, such as processors from Intel Corporation of Santa Clara, California and Motorola Corporation of

Schaumburg, Illinois. Such processors include, or may be in communication with, media, for example computer-readable media, which stores instructions that, when executed by the processor, cause the processor to perform the steps described herein.

Embodiments of computer-readable media include, but are not limited to, an electronic, optical, magnetic, or other storage or transmission device capable of providing a processor, such as the processor of client, with computer-readable

instructions. Other examples of suitable media include, but are not limited to, a floppy disk, CD-ROM, DVD, magnetic disk, memory chip, ROM, RAM, an ASIC, a configured processor, all optical media, all magnetic tape or other magnetic media, or any other medium from which a computer processor can read instructions. Also, various other forms of computer-readable media may transmit or carry instructions to a computer, including a router, private or public network, or other transmission device or channel, both wired and wireless. The instructions may comprise code from any suitable computer-programming language, including, for example, C, C++, C#, Visual Basic, Java, Python, Perl, Swift, Ruby, Unix, and JavaScript.

Computers are connected in some embodiments to a network or, in some embodiments, can be stand-alone machines. Computers may also include a number of external or internal devices such as a mouse, a CD-ROM, DVD, a keyboard, a display, or other input or output devices. Examples of computers are personal computers, digital assistants, personal digital assistants, cellular phones, mobile phones, smart phones, pagers, digital tablets, laptop computers, internet appliances, and other processor-based devices. In general, the computer-related to aspects of the technology provided herein may be any type of processor-based platform that operates on any operating system, such as Microsoft Windows, Linux, UNIX, Mac OS X, etc., capable of supporting one or more programs comprising the technology provided herein. All such components, computers, and systems described herein as associated with the technology may be logical or virtual.

Example 1 - temperature control

During the development of embodiments of the technology provided herein, experiments were conducted to test the temperature of a sample processed with an embodiment of the ultrasonic device. Samples were processed with an ultrasonic device as described herein and as shown in Figure 1. The device comprised a 1.7-MHz piezoelectric transducer with an active center. An external power supply provided electric current and/or voltage to the piezoelectric transducer and thermoelectric component. The experimental set-up also comprised a frequency controller and a cooling body in thermal communication with the thermoelectric component. A biological sample was processed with the ultrasonic device for 10 minutes.

A temperature sensor (e.g., a PT100 temperature sensor) was used to monitor the temperature of the sample and a second temperature sensor (e.g., a PT1000) was used to monitor the temperature of the piezoelectric transducer. The piezoelectric transducer was surrounded by FR4 insulating rings to provide thermal and vibrational isolation from the surroundings. The data collected show that the temperature of the piezoelectric transducer and of the sample remained below 75°C through the 10-minute

fragmentation time (Figure 2). Two independent ultrasonic units were tested.

Example 2 - transducer comprising a hole

During the development of embodiments of the technology provided herein, experiments were conducted to compare the fragmentation of nucleic acids using a piezoelectric transducer comprising a hole (see, e.g., Figure 3) with a standard piezoelectric transducer that does not have a hole. In particular, some embodiments of the technology are related to producing nucleic acid fragments of approximately 250 to 1000 base pairs for sequencing technologies. Initial experiments showed that adding water at the interface between the ultrasound transducer and the cartridge resulted in producing smaller nucleic acid fragments by improving coupling of the ultrasonic device to the sample chamber. However, the introduction of a coupling agent (e.g., a coupling gel or other liquid such as water) between the transducer and sample chamber is not desired or appropriate for certain embodiments of the technology such as those relating to processing a sample in a microfluidic cartridge.

Thus, to provide coupling between the piezoelectric transducer and a microfluidic cartridge comprising a biological sample without using a coupling agent (e.g., a coupling gel a coupling liquid or water) between the microfluidic cartridge and transducer, a hole was placed in the middle of the transducer (see, e.g., Figure 3). In addition, the experiment used a non-focusing transducer to provide efficient mixing of the sample and to provide random shearing and fragmentation of the nucleic acids in the sample.

Further, the piezoelectric transducer is spring mounted to provide a firm and flat contact between the cartridge and the piezoelectric transducer to provide adequate coupling.

Data were collected to compare nucleic acid fragmentation using a standard flat piezoelectric transducer with no hole and a flat piezoelectric transducer comprising a hole. The input nucleic acid had a peak in the size distribution at approximately 6000 base pairs (see, e.g., Figure 4, "Input WGA"). The standard flat piezoelectric transducer with no hole provided nucleic acid fragments having a peak in the size distribution at approximately 5000 base pairs (Figure 4, "3x3 min US"). The ultrasonic device comprising a flat piezoelectric transducer comprising a hole according to embodiments of the technology described herein provided nucleic acid fragments having a peak in the size distribution at approximately 2200 base pairs (Figure 4, "9 min US piezo + hole"). Further, experiments using the flat piezoelectric transducer comprising a hole were more reproducible than experiments using the flat piezoelectric transducer without a hole.

Example 3 - fragmentation time

During the development of embodiments of the technology provided herein, experiments were conducted to test the effect of fragmentation time on the production of nucleic acid fragments according to the technology described herein. In particular, a series of experiments was conducted using a current of 0.3 amperes. Further, S. Tset_max temperature of 70°C and a Tset_min temperature of 50°C for the piezoelectric transducer were used. The T_Set_max temperature is the maximum temperature the piezoelectric transducer can reach (as measured by a temperature sensor) before the power to the piezoelectric transducer is switched off to initiate a period of cooling for the piezoelectric transducer an/or for the sample. Then, when piezoelectric transducer reaches the T_set_min temperature, the power to the piezoelectric transducer is turned on until the

piezoelectric transducer reaches the T set_max temperature again.

The data collected show that the maximum of the fragment length distribution decreased as a function of increasing time up to a fragmentation time of 10 minutes (Figure 5). At fragmentation times greater than 10 minutes the maximum of the fragment length distribution was not further reduced (Figure 5).

Example 4 - current

During the development of embodiments of the technology provided herein, experiments were conducted to test the effect of current on the production of nucleic acid fragments according to the technology described herein. In particular, a series of experiments was conducted to assess the fragmentation of nucleic acids using an ultrasonic device supplied with currents ranging from 0.2 to 0.5 amperes.

The data showed that similar fragment sizes were produced with currents of 0.2,

0.3, 0.35, 0.4, and 0.45 amperes (Figure 6).

Parallel experiments were conducted to monitor the temperature of the sample at these currents. Data collected showed that a current of 0.3 amperes heated the ultrasonic device to approximately 50°C during a 10-minute fragmentation time. A current of 0.35 to 0.4 amperes heated the ultrasonic device to approximately 60 to 65°C during a 10-minute fragmentation time. As experiments showed that the sample temperature is greater than the temperature of the piezoelectric transducer (see, e.g., Figure 2), experiments were further conducted to monitor the temperature of the ultrasonic device (e.g., the piezoelectric transducer) and the temperature of the sample over a 10-minute fragmentation time (see Figure 7). The data showed that a current of 0.3 amperes produced a temperature of approximately 70°C in the sample after approximately 10 minutes of fragmentation time (Figure 7). To prevent samples from reaching temperatures greater than 70°C or more which can cause degradation of nucleic acids, a current of 0.3 amperes was chosen as the preferred current to use for the embodiment tested.

Example 5 - duty cycle

During the development of embodiments of the technology provided herein, experiments were conducted to test the effect of duty cycle on sample temperature. In particular, the effect of the duty cycle was investigated by measuring the fragmentation of nucleic acids after a lOminute fragmentation time over a range of T_set_max temperatures. Decreasing the duty cycle reduces the fraction of time that electric current is supplied to the piezoelectric transducer and thus reduces the fraction of time that the device is "on" over the lOminute fragmentation time.

Data collected showed that decreasing the T_Set_max temperature produces fragments having an increased maximum in the fragment size distribution (Figure 8). Further, lower T_set_max temperatures are associated with more error in the experiments (e.g., more variation and less reproducibility between experiments). See Figure 8. Example 6 - insulation ring

During the development of embodiments of the technology provided herein, experiments were conducted to test the thermal isolation of the ultrasonic device. In particular, the thermoelectric component was used to heat the device and infrared images were acquired to assess the temperature of the device and its surroundings. Data collected showed that the high temperatures were concentrated directly around the ultrasonic device. Further, the temperatures of adjacent components insulated from the heat source by the insulation ring were near ambient room temperature or slightly elevated (e.g., 20°C to 25°C).

All publications and patents mentioned in the above specification are herein incorporated by reference in their entirety for all purposes. Various modifications and variations of the described compositions, methods, and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in connection with specific exemplary embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various

modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims.

Claims

A device for producing nucleic acid fragments from a biological sample comprising nucleic acid, the device comprising:

a) a non-focusing ultrasonic transducer comprising a hole; and

b) a thermoelectric component attached to the non-focusing ultrasonic

transducer.

The device of claim 1 further comprising an insulating ring surrounding the ultrasonic transducer.

The device of claim 2 wherein the insulating ring comprises a heat resistant material.

The device of claim 2 wherein the insulating ring comprises a resin-reinforced, glass-reinforced, or polymer-reinforced laminate material.

The device of claim 2 wherein the insulating ring comprises an FR4 material.

The device of claim 1 wherein the thermoelectric component comprises a polymer coating

The device of claim 1 wherein the thermoelectric component comprises a PTFE coating, a perfluoroalkoxy (PFA) coating, a fluorinated ethylene propylene (FEP) coating, a polychlorotrifluoroethylene coating, a tetrafluorethylene- perfluoropropylene coating, a polyether ether ketone (PEEK) coating, or a nylon coating.

The device of claim 1 wherein the ultrasonic transducer is a piezoelectric transducer.

The device of claim 1 wherein the ultrasonic transducer is an electromagnetic transducer or a capacitive transducer.

10. The device of claim 1 wherein the thermoelectric component is a Peltier type thermoelectric component.

11. The device of claim 1 further comprising a heat sink to remove thermal energy from the device.

12. The device of claim 1 wherein the ultrasonic transducer is a 1-1000 MHz transducer.

13. The device of claim 1 wherein the ultrasonic transducer is a 1-100 MHz

transducer.

14. The device of claim 1 wherein the ultrasonic transducer is a 1-10 MHz

transducer.

15. The device of claim 1 further comprising a temperature sensor.

16. The device of claim 1 further comprising a temperature sensor to provide feedback control to the thermoelectric component.

17. The device of claim 1 wherein the thermoelectric component maintains the temperature of a sample below 99°C.

18. The device of claim 1 wherein the thermoelectric component maintains the temperature of a sample below 90°C.

19. The device of claim 1 wherein the thermoelectric component maintains the temperature of a sample below 85°C.

20. The device of claim 1 wherein the thermoelectric component maintains the temperature of a sample below 80°C.

21. The device of claim 1 wherein the thermoelectric component maintains the temperature of a sample below 75°C.

The device of claim 1 wherein the device produces nucleic acid fragments of 250 to 1000 base pairs.

The device of claim 1 wherein the device produces nucleic acid fragments of 1000 to 3000 base pairs.

The device of claim 1 wherein the device produces nucleic acid fragments of 1 to 20 kilobase pairs.

The device of claim 1 adapted for dry coupling to a vessel containing the sample.

A system for producing nucleic acid fragments from a biological sample comprising nucleic acid, the system comprising a plurality of devices according to claim 1.

The system of claim 26 wherein a first device is configured to lyse cells and a second device is configured to fragment nucleic acids.

The system of claim 26 comprising an interface to accept a microfluidic cartridge.

The system of claim 28 comprising a microfluidic cartridge comprising a cell lysis chamber and a nucleic acid fragmentation chamber.

The system of claim 26 comprising a spring member or vacuum to hold a device tightly against a cell lysis chamber.

The system of claim 26 comprising a spring or a vacuum to hold a device tightly against a nucleic acid fragmentation chamber.

The system of claim 26 further comprising a sensor to monitor cell lysis and/or a sensor to monitor nucleic acid fragmentation.

The system of claim 29 wherein the cell lysis chamber and/or the nucleic acid fragmentation chamber comprise(s) a vent.

34. The system of claim 29 wherein the cell lysis chamber and/or the nucleic acid fragmentation chamber comprise(s) a HEPA filter.

35. A method for producing nucleic acid fragments from a biological sample

comprising nucleic acid, the method comprising:

a) providing a device according to claim Y,

b) lysing cells in the biological sample using ultrasonic energy to produce a lysatei and

c) fragmenting nucleic acids in the lysate using ultrasonic energy to produce an output sample comprising fragmented nucleic acids.

36. The method of claim 35 further comprising monitoring the temperature of the biological sample, the lysate, and/or the output sample.

37. The method of claim 35 comprising providing electric current and/or electric

voltage to the device or apparatus when the temperature of the biological sample, the lysate, and/or the output sample is below a maximum temperature.

38. The method of claim 37 wherein the maximum temperature is 70 to 80°C.

39. The method of claim 37 comprising providing electric current and/or electric

voltage to the device for 10 minutes or less.

40. The method of claim 35 wherein the ultrasonic energy has a frequency of 1 to 100 MHz.

41. The method of claim 35 comprising cooling the device, apparatus, or sample with a thermoelectric component.

42. The method of claim 35 further comprising outputting the output sample to a NGS apparatus.

43. The method of claim 35 comprising providing a current of 0.1 to 0.5 amperes to an ultrasonic transducer of the device or apparatus.

44. The method of claim 35 comprising dry coupling the device or apparatus to a sample chamber holding the biological sample.

45. The method of claim 35 comprising holding the device or apparatus to a sample chamber using a spring or a vacuum.

46. The method of claim 35 comprising providing a microfluidic cartridge comprising the biological sample.

47. The method of claim 35 comprising inserting a microfluidic cartridge comprising the biological sample into an interface of the apparatus adapted to receive the microfluidic cartridge.

48. The method of claim 35 comprising providing a microfluidic cartridge comprising the biological sample and beads to cavitate and/or mix the sample.

49. The method of claim 35 comprising providing a microfluidic cartridge comprising the biological sample and grinding beads.

50. The method of claim 35 comprising providing a microfluidic cartridge comprising the biological sample and hardened steel beads, stainless steel beads, tungsten carbide beads, agate beads, zirconium oxide beads, PTFE beads, yttrium stabilized zircon beads, glass beads, or silica beads.

51. The method of claim 35 comprising providing a microfluidic cartridge comprising the biological sample and grinding beads having a diameter of approximately 2 mm.

52. The method of claim 35 wherein the output sample comprises nucleic acid

fragments of 250 to 1000 base pairs.

53. The method of claim 35 wherein the output sample comprises nucleic acid

fragments of 1000 to 3000 base pairs.

The method of claim 35 wherein the output sample comprises nucleic acid fragments of 1 to 20 kilobase pairs.

The method of claim 35 comprising providing a microfluidic cartridge comprising: a) one or more sample chambers connected to a depressurization chamber by a vented conduit comprising an aerosol filter; and

b) the biological sample.

The method of claim 54 wherein the aerosol filter is a HEPA filter.

A system for producing nucleic acid fragments from a biological sample comprising nucleic acid, the system comprising:

a) a microfluidic cartridge comprising a sample chamber to hold a biological sample; and

b) an apparatus comprising:

1) a non-focusing ultrasonic transducer comprising a hole to lyse cells and/or to fragment nucleic acids; and

2) a thermoelectric component attached to the non-focusing ultrasonic transducer to cool the ultrasonic transducer and/or to cool the biological sample.

The system of claim 57 further comprising a temperature sensor to monitor the temperature of the ultrasonic transducer and/or biological sample.

The system of claim 57 further comprising a microprocessor to receive

temperature data from a temperature sensor and to provide an electric current or voltage to the ultrasonic transducer.

The system of claim 57 further comprising a high-speed buffered digital input/output to provide a low ripple DC voltage to the thermoelectric component.

The system of claim 57 wherein the microfluidic cartridge comprises one or more sample chambers connected to a depressurization chamber by a vented conduit comprising an aerosol filter.

62. The system of claim 57 wherein the microfluidic cartridge comprises grinding beads.

63. The system of claim 57 wherein the microfluidic cartridge comprises hardened steel beads, stainless steel beads, tungsten carbide beads, agate beads, zirconium oxide beads, PTFE beads, yttrium stabilized zircon beads, glass beads, or silica beads.

64. The system of claim 57 configured to dry couple the microfluidic cartridge to the ultrasonic transducer.

65. The system of claim 57 wherein the sample chamber is vented.

66. The system of claim 57 wherein the sample chamber comprises a HEPA filter.

67. A system for sequencing a nucleic acid comprising the system of claim 57 and a NGS sequencing apparatus.