Hybridization capture

Abstract

The present disclosure provides a method for enriching a nucleic acid molecule.

Description

WSGR Reference No. 56252-721 .601HYBRIDIZATION CAPTURECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 718,304 filed on November 8, 2024, the entirety of which is incorporated herein by reference.BACKGROUND

[0002] Various DNA sequencing methods can employ various detection methods to determine the sequence of molecules in different types of DNA samples. For example, the input sample can be an isolation of many copies of the same genome, where the DNA is randomly sheared into smaller fragments (e.g., using hydrodynamic forces followed by further shearing with enzymatic fragmentation). For most sequencing technologies, exactly which molecules in a library are sequenced is random and the sequencing platform measures signal from a small proportion (e.g., < 1%) of the available molecules. By sequencing many copies of a single genome (e.g., usually in the range of 30-100 copies, i.e., 30-100X coverage), a consensus sequence can be determined.

[0003] Target enrichment is a molecular technique that can be used to reduce over-sequencing of portions of a DNA sample (e.g., portion of a genome) that are not of interest. Such methods selectively enrich DNA fragments of interest before sequencing, discarding molecules that are not of interest. This can be done by hybridization of single-stranded nucleic acids, called “probes”, to a targeted subset of the DNA molecules, physically pulling those targeted DNA fragments out of the mixture. Target enrichment significantly increases the sequencing coverage for regions of interest by selectively retaining targeted DNA fragments, thereby reducing the amount of excess sequencing coverage used on genomic regions not of interest. Such methods can enrich target regions 1,000’s of times relative to typical whole genome sequencing, reducing cost, increasing information content, and simplifying analysis.

[0004] For example, target enrichment can be applied in oncology for rare variant detection, biomarker detection, as well as in targeted screening panels for pathogen diagnostics. For example, oncology panels can be used to enrich DNA for sequencing small panels of known cancer genes, as in the KAPA™ HyperCap™ Oncology Panel, which provides probes for a combination of oncogenes including BRCA1, BRCA2, EGFR, TP53, and others.SUMMARY

[0005] Despite the widespread use of hybridization enrichment methods in DNA sequencing applications, significant opportunities for improvement exist. For instance, existing methods rely on manual human labor to perform several operations in sequence. This drives of the cost of the procedure, as well as reduces performance by introducing variability into the process. TheWSGR Reference No. 56252-721 .601 present disclosure fulfills a need for improved hybridization systems and methods that are automated, which can allow each person to process many more samples at a time. Less human intervention also improves the consistency of the method, resulting in more efficient use of reagents, higher and more reproducible yield, and ultimately higher quality sequencing data.

[0006] The methods described herein can be performed on various devices for droplet-based processing of biological samples. Such devices and applications may entail the manipulation of the droplets or magnetic particles contained in the droplets. The systems and methods described herein can use electrowetting on a dielectric (EWOD) technology to perform various fluidic manipulations in a single device, thereby automating previously labor-intensive hybridization procedures. Described herein are certain improvements that result in more efficient and reliable DNA hybridization and enrichment.

[0007] An aspect of the instant disclosure is a method for enriching a nucleic acid molecule, the method comprising: (a) mixing (i) a nucleic acid sample comprising a target nucleic acid molecule having a target sequence with (ii) a probe having a nucleic acid sequence complimentary to the target sequence to hybridize the probe with the target nucleic acid molecule, wherein said mixing is performed at a mixing temperature that is within about 5% of a mixing set-point temperature; (b) binding the probe to a solid support while under continuous mixing, wherein said binding is performed at a binding temperature that is within about 5% of a binding set-point temperature; (c) washing the probe bound to the solid support to discard portions of the nucleic acid sample that are not hybridized with the probe, wherein said washing is performed at a washing temperature that is within about 5% of a washing set-point temperature; and (d) eluting the target nucleic acid molecule from the washed probe, thereby enriching the target nucleic acid molecule relative to the nucleic acid sample. In some embodiments, (a)-(d) are performed on an electrowetting on dielectric (EWOD) device. In some embodiments, (a) and (b) are performed concurrently. In some embodiments, (a)-(b) are completed in less than about 30 minutes. In some embodiments, (a)-(d) are completed in less than about 60 minutes. In some embodiments, the target molecule is eluted in a PCR master mix. In some embodiments, the method further comprises amplifying the eluted target molecule. In some embodiments, the method further comprises sequencing the eluted target molecule. In some embodiments, the method further comprises sequencing the target molecule. In some embodiments, the probe is biotinylated. In some embodiments, the probe is bound to a streptavidin solid support. In some embodiments, the solid support is magnetic. In some embodiments, the solid support is aggregated using a magnet. In some embodiments, (b) is commenced within 10 seconds of the conclusion of mixing; or (c) is commenced within 10 seconds of the conclusion of binding; or (d) is commenced within 10 seconds of the conclusionWSGR Reference No. 56252-721 .601 of washing; or any combination thereof. In some embodiments, the nucleic acid sample comprises a plurality of molecules having the target sequence. In some embodiments, a plurality of nucleic acid samples are processed in parallel with the nucleic acid sample on an EWOD device. In some embodiments, the solid support has a plurality of copies of the probe attached thereto. In some embodiments, mixing is performed in a droplet of fluid on a surface of an EWOD device in proximity to an array of electrodes. In some embodiments, the droplet is subsequently transferred to a portion of the EWOD device not in proximity to the array of electrodes. In some embodiments, (a) forms a mixture of nucleic acid molecules comprising fragments of genomic DNA. In some embodiments, the fragments are prepared by mechanical and / or enzymatic shearing. In some embodiments, the fragments have adapters ligated thereto, wherein the adapters facilitate sequencing of the fragments. In some embodiments, hybridization is performed for at least about 4-22 hours. In some embodiments, hybridization is performed at a set-point temperature of about 55 °C, about 65 °C, or about 75 °C. In some embodiments, any reagents that are added are pre-warmed to a set-point temperature. In some embodiments, one or more consumables are utilized during (a)-(d), and wherein at least one of the one or more consumables is pre-warmed to a set-point temperature. In some embodiments, the one or more consumables comprises a fluidic tip or beads. In some embodiments, the method further comprises performing (c) a plurality of times. In some embodiments, the bound probe is washed with a first wash buffer at 65°C. In some embodiments, the method further comprises washing the bound probe twice with a stringent wash buffer at 65°C. In some embodiments, the method further comprises washing the bound probe with the first wash buffer at room temperature. In some embodiments, the method further comprises washing the bound probe with a second wash buffer at room temperature. In some embodiments, the method further comprises washing the bound probe with a third wash buffer at room temperature. In some embodiments, sequencing the eluted target molecule is achieved with a fold-80 penalty of less than about 1.5, wherein the fold-80 penalty is an amount of additional sequencing coverage required to bring 80% of the sequenced bases up to an average coverage. In some embodiments, sequencing the eluted target molecule is achieved with an on-target base percentage of greater than about 50% when the sample is derived from preserved tissue, wherein the on-target base percentage is a proportion of the bases sequenced that are aligned to the target sequence. In some embodiments, sequencing the eluted target molecule is achieved with an on-target base percentage of greater than about 85% when the sample is derived from cfDNA, wherein the on-target base percentage is a proportion of the bases sequenced that are aligned to the target sequence. In some embodiments, sequencing the eluted target molecule is achieved with an on-target base percentage of greater than about 90% when the sample is derived from genomic DNA, wherein the on-target baseWSGR Reference No. 56252-721 .601 percentage is a proportion of the bases sequenced that are aligned to the target sequence. In some embodiments, the target nucleic acid molecule is enriched at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, or at least about 1,000-fold relative to an initial concentration of the target nucleic acid molecule in the sample. In some embodiments, a plurality of target sequences is enriched using (a)-(d). In some embodiments, the plurality of target sequences corresponds to an oncogene panel comprising the BRCA1, BRCA2, EGFR, and TP53 genes. In some embodiments, the plurality of target sequences corresponds to a panel for detection of pathogens, characterizing drug metabolism, or characterizing patient therapeutic response.

[0008] In some embodiments, the mixing set-point temperature, the binding set-point temperature, and the washing set-point temperature are each within about 5% of a common setpoint temperature. In some embodiments, the solid support, a hybridization buffer, a washing buffer, and / or an elution buffer are warmed to the common set-point temperature prior to being used in the method.

[0009] In some embodiments, the mixing set-point temperature, the binding set-point temperature, and the washing set-point temperature are each within about 5% of a common setpoint temperature.

[0010] In some embodiments, (b) is for at least 15 minutes.

[0011] In some embodiments, the solid support, a hybridization buffer, a washing buffer, and / or an elution buffer are warmed to the common set-point temperature prior to being used in the method.

[0012] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:WSGR Reference No. 56252-721 .601

[0014] FIG. 1 shows an example of a schematic detailing a library pool of varying products, a probe to DNA-target hybridization, a bead capture reaction, wash steps, and finally a final library comprising the desired DNA regions, according to the systems and methods described herein.

[0015] FIG. 2 shows an example of equipment required to process samples manually, according to prior systems and methods.

[0016] FIG. 3 shows a prophetic example of temperature fluctuations in a manual workflow, according to prior systems and methods.

[0017] FIG. 4 shows a prophetic example of the impact of temperature variation during wash steps on quality metrics, according to the systems and methods described herein.

[0018] FIG. 5 shows an example of an electrowetting on dielectrode (EWOD) device, according to the systems and methods described herein.

[0019] FIG. 6 shows an example of a top-down view of a reaction surface on an EWOD device, according to the systems and methods described herein.

[0020] FIG. 7 shows an example of a top-down view of a reaction surface on an EWOD device with a droplet undergoing side-to-side fluid mixing (A) and mechanical mixing (B), according to the systems and methods described herein.

[0021] FIG. 8 shows an example of waste removal from the reaction surface from an initial time (A) to a later time (B), according to the systems and methods described herein.

[0022] FIG. 9 shows an example of a prophetic graph of sample temperature that is consistent through the process steps and near the set-point temperature, according to the systems and methods described herein.

[0023] FIG. 10 shows an example of a computer system configured to control an EWOD device, according to the systems and methods described herein.

[0024] FIG. 11 shows an example of an experimental design to compare the methods described herein with a manual hybridization method.

[0025] FIG. 12 shows an example of post-capture yields, comparing to the systems and methods described herein with a manual hybridization method.

[0026] FIG. 13 shows an example of mean target coverage, comparing to the systems and methods described herein with a manual hybridization method.

[0027] FIG. 14 shows an example of fold enrichment, comparing to the systems and methods described herein with a manual hybridization method.

[0028] FIG. 15 shows an example of fold-80 penalty, comparing to the systems and methods described herein with a manual hybridization method.WSGR Reference No. 56252-721 .601

[0029] FIG. 16 shows an example of PCR duplicates, comparing to the systems and methods described herein with a manual hybridization method.

[0030] FIG. 17 shows an example of on-target rates, comparing to the systems and methods described herein with a manual hybridization method.

[0031] FIG. 18 shows an example of on-target rates, comparing to the systems and methods described herein with a manual hybridization method.

[0032] FIG. 19 shows an example of sequencing coverage for a difficult to capture region, comparing to the systems and methods described herein with a manual hybridization method.

[0033] FIG. 20 shows an example of multiplexing, comparing to the systems and methods described herein with a manual hybridization method.DETAILED DESCRIPTION

[0034] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0035] FIG. 1 shows an example of a hybridization enrichment method. Here, a pool of library molecules 100 is generated from DNA by enzymatic and / or mechanical fragmentation. In some cases, the library of molecules are cell-free DNA (e.g., cfDNA, circulating in the blood stream). Various other DNA samples are known to those skilled in the art. In some cases, the molecules have platform-specific sequencing adapters ligated thereto. The sample contains DNA segments of interest (black) and not of interest (gray).

[0036] After library molecules are generated, biotinylated single-stranded nucleic acid “probes” 102 that are complementary to the target regions, are added to hybridize to DNA fragments. Any DNA fragment hybridized to the biotinylated probes can be pulled out of solution 104 using Streptavidin magnetic beads, while unbound, untargeted DNA fragments will remain in solution 110. One or more washes 106 can be performed to remove any DNA fragments that are not strongly hybridized 112 to the probes (here depicted as some non-specific binding between a probe and a non-target DNA). The final product is an enriched library 108 of targeted DNA.

[0037] FIG. 2 shows an example of equipment required to process samples manually. The diagram depicts the equipment required for a manual target enrichment workflow, broken down by each step. The library 200 input into the workflow can be mechanically or enzymatically sheared DNA, and is followed by hybridization 202, bead capture 204, and washing 206, and elution 208. Note that wash steps are typically performed two or more times. The workflow hasWSGR Reference No. 56252-721 .601 three steps that are sensitive to temperature (hybridization, bead capture, and washing). To carry out this workflow manually, there are several pieces of equipment required that a person must quickly transfer tubes between: thermocycler, vortexer, centrifuge, multi-channel pipettors, and magnetic bead racks.

[0038] An example manual workflow is as follows. During hybridization, the user mixes probes with the libraries and incubates them for 16-20 hours at 55°C in a thermocycler. During bead capture, the user must remove the hybridization reactions from the thermocycler, add the capture beads, and return to the thermocycler at 55°C to incubate for several minutes.

[0039] Washing can be the most temperature sensitive step, as the temperature drives the hybridization strength between probe and target. Wash buffer must be pre-warmed in a thermocycler before adding to the bead capture reaction. After adding a washing buffer, the person must quickly vortex the reaction to ensure a homogenous mixture, centrifuge quickly, and return to a thermocycler to incubate for several minutes. The beads are then pelleted on a magnetic rack at room temperature, and this washing process is repeated several times. The user must act very quickly to reduce the impact of vortexing, centrifugation, and bead pelleting at room temperature on the reaction temperature because it is a main driver of selectively for the target DNA.

[0040] The prior manual hybridization methods can suffer from several problems. There are several metrics to consider when evaluating a target enrichment sequencing experiment including the Fold-80 penalty, percent off-target (or on-target) bases, and zero coverage bases. The Fold-80 penalty is the amount of additional sequencing coverage required to bring 80% of the target bases up to the average coverage. For example, if all target bases have the same fold coverage, the Fold-80 penalty will be 1. A Fold-80 penalty score between 1.3-1.6 is considered good, indicating that most target regions have mostly consistent sequencing coverage. Percent off-target bases is the proportion of off-target library molecules sequenced, as determined by aligning all sequencing reads against the genome. Similarly, percent on-target bases is the proportion of on-target library molecules sequenced, as determined by aligning all sequencing reads against the genome. Finally, Zero coverage targets are the targeted regions that resulted in no sequencing coverage.

[0041] FIG. 3 shows a prophetic line graph depicting the many temperature fluctuations throughout a manual target enrichment workflow. Performing this workflow manually requires moving the reagents from the thermocycler to room temperature to add reagents, vortex, centrifuge, and pellet on a magnetic tube rack. By transferring the tubes from thermocycler to vortexer, and centrifuging, variations in temperature are introduced. Since temperature is aWSGR Reference No. 56252-721 .601 driver of several quality metrics, the quality of the targeted enrichment sequencing experiment is impacted, depending on the skill level of the operator.

[0042] FIG. 4. shows a prophetic example of the impact of temperature variation during wash steps on quality metrics. Temperature, particularly during the wash steps, is a critical driver of target enrichment success that impacts all of the quality metrics described above. Here, a setpoint temperature 400 is shown where quality metrics are optimized. A lower temperature than the set-point during washing stabilizes hybridization, which can lead to off-target fragments being retained during enrichment. This will increase the proportion of off-target bases, reducing the relative enrichment of the target regions. Conversely, a higher temperature than the set-point reduces the stability of hybridized probes to targets, which can cause a loss of target regions that are not as tightly bound as others (for example, regions with lower GC content), introducing GC bias. Overall, temperature is a key determinant of the amount of off-target reads, evenness of sequencing coverage across target regions (represented by increased Fold-80 penalty), and can impact how many targeted regions have zero coverage.

[0043] GC bias can be introduced into the sequencing run if the temperature is not consistent due to the different relative strengths of G-C bonds vs. A-T bonds. This can result in regions with lower G-C bonds to be removed during the stringent wash steps, negatively impacting the performance of the panel.

[0044] Similar to GC bias, there can be off-target molecules enriched due to probes hybridizing weakly with similar but non-targeted DNA sequences. A lower temperature during the stringent wash steps will result in a higher proportion of off-target reads since the lower temperature helps stabilize the hybridization reaction between loosely bound probes.

[0045] Among the various biological issues that are problematic with current manual workflows, there is the vast complexity of tools and equipment needed to perform a single workflow (see, FIG. 2). A tedious workflow adds additional points of error for a researcher that then amplify with increased throughput.Electrowetting devices

[0046] Described herein is a method for target enrichment of nucleic acids using an electrowetting based system. The electrowetting system can be configured to provide fluidic operations including motion, mixing, holding, and splitting liquid from magnetic particles. The electrowetting-on-dielectric (EWOD) module enables multiple droplets to be held and move on the surface simultaneously. Referring to FIG. 5, this system can include four modules: heating 500, magnetic 502, mixing 504, and dispensing 506. Together, these modules provide a unique environment that enables greater precision in controlling the biological reactions occurring onWSGR Reference No. 56252-721 .601 the reaction surface than currently in the field. The EWOD module, or reaction surface, sits on top of the mixing module, which houses both the heating and magnetic modules. The dispenser module is located above the mixing module, able to deliver reagents directly to the reaction surface. Additional description of suitable EWOD devices can be found in U.S. Patent Serial No. 11,123,729, U.S. Patent Application Serial No. 18 / 061,133, U.S. Patent Application Serial No. 18 / 462,365, U.S. Patent Application Serial No. 18 / 628,148, PCT Patent Application Serial No. US2023 / 068,630, and PCT Patent Application Serial No. US2023 / 068,253, each of which is incorporated by reference herein in its entirety.

[0047] Described herein is a device where temperature can be precisely controlled across the reaction surface. This includes control over long incubation steps, as well as control during transitions between biological steps and reactions. This module provides the opportunity to allow distinct liquid droplets to be acclimated to the same temperatures before combining for a given biological reaction. This helps to mitigate any temperature fluctuations that can occur during dispensing. Quick and even heating can provide more efficient reaction zones for liquids on the device. In addition, reagent vessels utilize temperature control to ensure liquids are at appropriate temperature before being added to the reaction surface.

[0048] FIG. 6 shows an example of a top-down view of a reaction surface on an EWOD device, according to the systems and methods described herein. The bead 600 and supernatant 602 can be separated upon magnet activation. Placement of the magnetic module within the system allows for direct interaction with the reaction surface. The magnetic module can operate below the reaction surface, allowing it to be operated in tandem with the heating module, enabling steps involving both mechanisms to occur simultaneously. This allows samples on the reaction surface to remain at a desired set temperature when suspended in liquid or concentrated after supernatant removal. After incubation, beads can be separated from the liquid supernatant upon activation of the magnet. This automated activation allows for temperature consistency throughout the duration of any bead removal step, which is not achieved with prior manual methods.

[0049] FIG. 7 shows an example of a top-down view of a reaction surface on an EWOD device with a droplet undergoing side-to-side fluid mixing (A) and mechanical mixing (B), according to the systems and methods described herein. The device can utilize multiple techniques to mix samples on the reaction surface. First, EWOD allows for the control of droplet movement 700 in both the vertical and horizontal axis (A), allowing for liquid mixing during otherwise static reactions. Second, the module can house an internal vibration mechanism. This mechanism can introduce mechanical vibrations 702 to liquids (B). Mechanical vibrations can accommodate these various liquid properties by tuning amplitude and frequency accordingly.WSGR Reference No. 56252-721 .601

[0050] The EWOD device can house a dispensing module. This dispenser can perform direct addition of samples from a reagent vessel to a specific zone on the reaction surface. This utility can enable multiple reactions to occur on a single surface, as well as control timing for both starting and ending multiple reactions in parallel through the use of the EWOD module across all samples.

[0051] FIG. 8 shows waste removal from the reaction surface from an initial time (A) to a later time (B). Specifically, the method can utilize EWOD to move liquid waste toward a sponge 800 for removal of liquid waste 802 from the reaction surface 804 (i.e., by soaking 806 into the sponge).

[0052] During bead washing steps, the systems and methods described herein reduce or eliminate temperature fluctuations caused by removal of the sample from a controlled thermocycler environment, or during addition and removal of wash buffers. This helps to maintain the high sensitivity of the reaction, allowing for more efficient bead washing. Removal of liquid waste is accomplished by multiple modules working in parallel including, EWOD and magnetic modules. Liquid waste can be separated from pelleted magnetic beads and moved into an absorbent waste removal medium, such as a sponge. This enables the device to perform the workflows without manual intervention, and with reduce use of pipette tips.

[0053] Described herein is a method to increase the efficiency of enrichment in a target enrichment workflow on an electrowetting system. The device described herein provides a platform integrated with various modules that improve the sensitivity and specificity of enrichment-based workflows through precise temperature control, improved mixing and washing conditions, waste removal, and efficient reaction performance. This results in a lower Fold-80 penalty, reduced off-target bases, and reduces zero-coverage bases.

[0054] In contrast to temperature fluctuation as shown for prior methods in FIG. 3, the methods and systems described herein can result in more consistent temperature control as shown in FIG.9. Here, constant temperature control can be achieved throughout reaction steps of the target enrichment reaction. During target enrichment and bead-based capture reactions, the reaction can be incubated at a desired set temperature 900 and mixed to drive binding efficiency. This is applicable to multiple steps in the target enrichment workflow. First, during probe hybridization 902 with target DNA, again during bead capture 904 via probe hybridization with magnetic beads, and lastly during subsequent bead washing 906 steps. Constant temperature during all of these steps helps to maximize the enrichment yield and minimize the time duration of the overall reaction. As all steps of the workflow are occurring on a single reaction surface, the reactions can be maintained at a constant temperature during mixing, bead pelleting, and movement.WSGR Reference No. 56252-721 .601

[0055] Using the systems and methods described herein increase the efficiency of hybridization capture in several ways. Due to reduced time spent transferring liquids between tubes, the total reaction time is decreased. Due to the ability to utilize constant mixing, typical stationary, intube, reactions can have reduced incubation times. With the entirety of the target enrichment protocol happening on a single reaction surface, the methods reduce or eliminate the need to transfer samples between tubes and the need for pipet mixing, removing the sample loss that normally occurs during these steps manually. Upon completion of the reaction, samples can be collected directly from the surface without the use of intermediate tubes.

[0056] There are several ways to increase the efficiency of enrichment in a target enrichmentbased workflow on an electrowetting system. The EWOD device described herein can provide a platform integrated with various modules that can be independently modified or changed, depending on the application.

[0057] For example, a heated liquid transfer mechanism can be used. In some embodiments, a liquid handling device can be used that has a mechanism to keep liquids at a given temperature inside a pipette tip. For example, the tip can be heated by resistive heating of the tip itself. The tip can also be seated inside a heater with heat transfer happening either via conduction or convection.

[0058] For example, nucleic acid quantification can be used. In some embodiments, a liquid handling device can be used that has a mechanism to quantify total output yield on the surface of the device. For example, using a fluorometric quantification method detected through additional sensors on the device.

[0059] For example, nucleic acid yield normalization can be used. In some embodiments, a liquid handling device can be used that has a mechanism to normalize the total output yield across samples without the need for additional measurements. For example, using saturating nucleic acid-binding beads, or an enzymatic reaction to digest excess library on the surface of the device will normalize the final yield.

[0060] For example, nucleic acid size selection can be used. In some embodiments, a liquid handling device can be used that has a mechanism to select for a specific range of DNA fragment lengths through the use of proprietary buffers, mixtures, or nucleic acid-binding beads without the need for additional steps.

[0061] For example, nucleic acid denaturing can be used. In some embodiments, a liquid handling device can be used that has a mechanism to convert double-stranded DNA libraries to single-stranded DNA libraries by the addition of a proprietary buffer to the module.WSGR Reference No. 56252-721 .601

[0062] For example, nucleic acid amplification can be used. In some embodiments, a device that can amplify library output in an isothermal enzymatic reaction without additional heating requirements using enzymatic reactions can be used.

[0063] For example, a pluronic can be used to overcome bead repulsion allowing the beads to be pelleted well. Pluronics, also known as poloxamers, are a class of synthetic block copolymers which consist of hydrophilic poly(ethylene oxide) (PEO) and hydrophobic polypropylene oxide) (PPO), arranged in an A-B-A triblock structure.

[0064] The sample throughput can be high. In some embodiments, a device that can perform the reactions described herein in sample quantities in the amounts of 1, 4, 8, 12, 24, 96, 384, or more at a time. Multiple samples can be processed on each reaction surface to further increase the sample volume.Hybridization Capture Method

[0065] In an aspect, provided herein is a method for enriching a nucleic acid molecule. The method can include mixing (i) a nucleic acid sample comprising a target nucleic acid molecule having a target sequence with (ii) a probe having a nucleic acid sequence complimentary to the target sequence. The mixing can be performed such that the probe hybridizes with the target nucleic acid molecule, where the mixing is performed at a mixing temperature that is within about 5% of a mixing set-point temperature.

[0066] In some embodiments, the mixing is performed at a mixing temperature that is within about 10%, within about 8%, within about 5%, within about 3%, within about 1%, within about 0.5%, or within about 0.1%, of a mixing set-point temperature.

[0067] The method can further include binding the probe to a solid support for at least 15 minutes while under continuous mixing. Wherein the binding is performed at a binding temperature that is within about 5% of a binding set-point temperature.

[0068] In some embodiments, the mixing is performed at a binding temperature that is within about 10%, within about 8%, within about 5%, within about 3%, within about 1%, within about 0.5%, or within about 0.1%, of a binding set-point temperature.

[0069] The binding can be performed for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 90 minutes, at least 120 minutes, or at least 180 minutes. In some embodiments, the binding can be performed for less than 15 minutes.

[0070] The method can further include washing the bound probe to discard portions of the nucleic acid sample that are not hybridized with the probe, wherein said washing is performed at a washing temperature that is within about 5% of a washing set-point temperature.WSGR Reference No. 56252-721 .601

[0071] In some embodiments, the washing is performed at a washing temperature that is within about 10%, within about 8%, within about 5%, within about 3%, within about 1%, within about 0.5%, or within about 0.1%, of a washing set-point temperature.

[0072] The method can further include eluting the target nucleic acid molecule from the washed probe, thereby enriching the target nucleic acid molecule relative to the nucleic acid sample.

[0073] In some embodiments, mixing (hybridization), binding, washing, and eluting are performed on an electrowetting on dielectric (EWOD) device.

[0074] In some cases, mixing (hybridization) and binding are performed concurrently.

[0075] The steps of the method can be performed rapidly. In some embodiments, mixing (hybridization) and binding are performed in less than about 5 minutes, less than about 15 minutes, less than about 30 minutes, less than about 45 minutes, less than about 60 minutes, less than about 90 minutes, or less than about 180 minutes. In some embodiments, mixing (hybridization), binding, washing, and eluting are performed in less than about 5 minutes, less than about 15 minutes, less than about 30 minutes, less than about 45 minutes, less than about 60 minutes, less than about 90 minutes, or less than about 180 minutes.

[0076] In some instances, the target molecule is eluted in a PCR master mix. In some embodiments, the method further includes amplifying the eluted target molecule. The method can further comprise sequencing the eluted target molecule.

[0077] The probe can be biotinylated. The probe can be bound to a streptavidin solid support. The solid support can be magnetic. The solid support can be aggregated using a magnet.

[0078] The next step in the method can be commenced within a suitably short time of a prior step being completed. For instance, binding can be commenced within 1 second, within 5 seconds, within 10 seconds, or within 30 seconds of the conclusion of mixing (hybridization). Washing can be commenced within 1 second, within 5 seconds, within 10 seconds, or within 30 seconds of the conclusion of binding. Eluting can be commenced within 1 second, within 5 seconds, within 10 seconds, or within 30 seconds of the conclusion of washing.

[0079] The nucleic acid sample can comprise a plurality of molecules having the target sequence. A plurality of nucleic acid samples can be processed in parallel on an EWOD device. In some cases, the solid support has a plurality of copies of the probe attached thereto.

[0080] The mixing can be performed in a droplet of fluid on a surface of an EWOD device in proximity to an array of electrodes. The droplet can be subsequently transferred to a portion of the EWOD device not in proximity to the array of electrodes.WSGR Reference No. 56252-721 .601

[0081] The mixture of nucleic acid molecules can be fragments of genomic DNA. The fragments can be prepared by mechanical and / or enzymatic shearing. The fragments can have adapters ligated thereto, which adapters facilitate sequencing of the fragments.

[0082] Hybridization can be performed for at least about 1, at least about 2, at least about 4, at least about 8, at least about 12, at least about 16, at least about 22, or at least about 24 hours.

[0083] The hybridization can be performed at a set-point temperature of about 55 °C, about 65 °C, or about 75 °C.

[0084] In some cases, any reagents that are added are pre-warmed to a set-point temperature. Similarly, any consumables such as fluidic tips or beads can be pre-warmed to a set-point temperature.

[0085] The method can comprise washing the bound probe a plurality of times. The bound probe can be washed with a first wash buffer at 65°C. The method can further comprise washing the bound probe twice with a stringent wash buffer at 65°C. The method can further comprise washing the bound probe with the first wash buffer at room temperature. The method can further comprise washing the bound probe with a second wash buffer at room temperature. The method can further comprise washing the bound probe with a third wash buffer at room temperature.

[0086] In some embodiments, the mixing set-point temperature, the binding set-point temperature, and the washing set-point temperature are each within about 5% of a common setpoint temperature. The common set-point temperature can be about 55 °C, about 65 °C, or about 75 °C. In some embodiments, the mixing, binding, and / or washing are performed at a temperature that is within about 10%, within about 8%, within about 5%, within about 3%, within about 1%, within about 0.5%, or within about 0.1%, of the common set-point temperature.Computer systems

[0087] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 10 shows a computer system 1001 that is programmed or otherwise configured to manipulate droplet operations. The computer system 1001 can regulate various aspects of the present disclosure, such as, for example, controlling vibrations and / or vibration-assisted mixing, controlling a magnetic field with respect to a droplet provided on the surface of an array, controlling electrode activation, or controlling the power source to the electrowetting device. The computer system 1001 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.WSGR Reference No. 56252-721 .601

[0088] The computer system 1001 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 1005, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 1001 also includes memory or memory location 1010 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 1015 (e.g., hard disk), communication interface 1020 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1025, such as cache, other memory, data storage and / or electronic display adapters. The memory 1010, storage unit 1015, interface 1020 and peripheral devices 1025 are in communication with the CPU 1005 through a communication bus (solid lines), such as a motherboard. The storage unit 1015 can be a data storage unit (or data repository) for storing data. The computer system 1001 can be operatively coupled to a computer network (“network”) 1030 with the aid of the communication interface 1020. The network 1030 can be the Internet, an internet and / or extranet, or an intranet and / or extranet that is in communication with the Internet. The network 1030 in some cases is a telecommunication and / or data network. The network 1030 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 1030, in some cases with the aid of the computer system 1001, can implement a peer-to-peer network, which may enable devices coupled to the computer system 1001 to behave as a client or a server.

[0089] The CPU 1005 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1010. The instructions can be directed to the CPU 1005, which can subsequently program or otherwise configure the CPU 1005 to implement methods of the present disclosure. Examples of operations performed by the CPU 1005 can include fetch, decode, execute, and writeback.

[0090] The CPU 1005 can be part of a circuit, such as an integrated circuit. One or more other components of the system 1001 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0091] The storage unit 1015 can store files, such as drivers, libraries and saved programs. The storage unit 1015 can store user data, e.g., user preferences and user programs. The computer system 1001 in some cases can include one or more additional data storage units that are external to the computer system 1001, such as located on a remote server that is in communication with the computer system 1001 through an intranet or the Internet.

[0092] The computer system 1001 can communicate with one or more remote computer systems through the network 1030. For instance, the computer system 1001 can communicate with a remote computer system of a user. Examples of remote computer systems include personalWSGR Reference No. 56252-721 .601 computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 1001 via the network 1030

[0093] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1001, such as, for example, on the memory 1010 or electronic storage unit 1015. The machine executable or machine-readable code can be provided in the form of software. During use, the code can be executed by the processor 1005. In some cases, the code can be retrieved from the storage unit 1015 and stored on the memory 1010 for ready access by the processor 1005. In some situations, the electronic storage unit 1015 can be precluded, and machine-executable instructions are stored on memory 1010.

[0094] The code can be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.

[0095] Aspects of the systems and methods provided herein, such as the computer system 1001, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and / or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-WSGR Reference No. 56252-721 .601 transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0096] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0097] The computer system 1001 can include or be in communication with an electronic display 1035 that comprises a user interface (UI) 1040 for providing, for example, the patterns of droplet movement and / or electrode actuation described herein. Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0098] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 1005. The algorithm can, for example, optimize and execute the patterns of droplet movement and / or electrode actuation described herein.Example 1 - Gene panels

[0099] In some embodiments the plurality of target sequences correspond to an oncogene panel. The panel can comprise the BRCA1, BRCA2, EGFR, and TP53 genes.

[0100] In some cases, the plurality of target sequences correspond to an panel for detection of pathogens, characterizing drug metabolism, or characterizing patient therapeutic response.WSGR Reference No. 56252-721 .601

[0101] For example, the KAPA HyperCap Oncology panel is a 214 kilobase panel that targets a subset of cancer-related genes and coding sequences from the human genome for research purposes only. The targets are listed below.Table 1: KAPA Hyper Cap

[0102] Other examples are the Twist Biosciences PGx panel, which targets genes that are critical to drug metabolism and patient therapeutic response (like CYP2D6, HLA-A, HLA-B), in addition to the Twist Biosciences Alliance panels that provide panels for cancer gene methylation, clinical exome research, rare diseases, and hereditary oncology.Example 2 - Experimental design comparing present method with manual method

[0103] An objective of this example is to test variability in hybridization capture metrics between manual and the present workflows when performed by 3 different users. FIG. 11 shows an example of an experimental design to compare the methods described herein with a manual hybridization method. The experiment tests how efficiently the present workflow can handle multiplexing of 1-20 samples for each capture.

[0104] In Phase I: Three individual users set up both manual and automated workflows with four samples each for each workflow for hybridization capture using the exome panel. The samples captured were sequenced and analyzed for various hybridization capture metrics.WSGR Reference No. 56252-721 .601

[0105] In Phase II: Three individual users set up both manual and automated workflows with four samples each for each workflow for hybridization capture using the exome panel. The samples captured were sequenced and analyzed for various hybridization capture metrics.

[0106] In Phase III: One user performed an experiment for between 1-20 samples for exome capture. The captured samples were sequenced and analyzed for various hybridization capture metrics.

[0107] As used herein:• Chemistry: ID xGen™ Hybridization Capture workflow• Exome Panel: IDT xGen™ Exome v2 Hyb Panel• Pan-cancer panel: IDT xGen™ Pan-Cancer Hyb PanelExample 3 - Performance comparison of present method with manual method

[0108] The automated method for hybridization enrichment described herein shows excellent performance in comparison to prior manual methods (e.g., lacking precise temperature control). The experiment described in Example 2 was performed.

[0109] For example, FIG. 12 shows an example of post-capture yields, comparing to the systems and methods described herein with a manual hybridization method. In this depicted example, “Callisto 1,” “Callisto 2,” and “Callisto 3” all refer to uses of the systems and methods described herein, while “Manual 1,” “Manual 2,” and “Manual 3” all refer to uses of the previous system and methods as shown in FIG. 2. The present method delivers more consistent post-capture yields using the Exome and Pan Cancer panel with a minimum of 125 ng of total yield. This will result in more consistent coverage for operators of the methods described herein.

[0110] FIG. 13 shows an example of mean target coverage, comparing to the systems and methods described herein with a manual hybridization method. In this depicted example, “Callisto 1,” “Callisto 2,” and “Callisto 3” all refer to uses of the systems and methods described herein, while “Manual 1,” “Manual 2,” and “Manual 3” all refer to uses of the previous system and method as shown in FIG. 2. The method described herein delivers mean target coverage similar to manual with a minimum of 60X (50 million reads) coverage for Exome and 200X (5 million reads) coverage for Pan Cancer panels.[oni] FIG. 14 shows an example of fold enrichment, comparing to the systems and methods described herein with a manual hybridization method. The automated method outperforms manual with a fold enrichment greater than 50 for Exome and greater than 2500 for Pan Cancer. In this depicted example, “Callisto” refers to uses of the systems and methods described herein, while “Manual” refers to uses of the previous system and method as shown in FIG. 2. The presently described method results in more consistent fold enrichment for Pan Cancer panelWSGR Reference No. 56252-721 .601(e.g., because of the unique advantages of method with temperature control, efficient bead mixing and waste removal). In some embodiments, target nucleic acid molecule is enriched at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, or at least about 1,000-fold relative to an initial concentration of the target nucleic acid molecule in the sample.

[0112] FIG. 15 shows an example of fold-80 penalty, comparing to the systems and methods described herein with a manual hybridization method. In this depicted example, “Callisto 1,” “Callisto 2,” and “Callisto 3” all refer to uses of the systems and methods described herein, while “Manual 1,” “Manual 2,” and “Manual 3” all refer to uses of the previous system and method as shown in FIG. 2. The automated method described herein performs better than manual in uniformity of coverage compared to manual with fold-80 penalty less than 1.5 (e.g., because of the unique advantages with temperature control, efficient bead mixing and waste removal). In some instances, sequencing the eluted target molecule is achieved with a fold-80 penalty of less than about 1.5, wherein the fold-80 penalty is an amount of additional sequencing coverage required to bring 80% of the sequenced bases up to an average coverage.

[0113] FIG. 16 shows an example of PCR duplicates, comparing to the systems and methods described herein with a manual hybridization method. In this depicted example, “Callisto 1,” “Callisto 2,” and “Callisto 3” all refer to uses of the systems and methods described herein, while “Manual 1,” “Manual 2,” and “Manual 3” all refer to uses of the previous system and method as shown in FIG. 2. The method described herein performs similar to manual in duplication rates with PCR duplicates less than 10%.

[0114] FIG. 17 shows an example of on-target rates, comparing to the systems and methods described herein with a manual hybridization method. In this depicted example, “Callisto 1,” “Callisto 2,” and “Callisto 3” all refer to uses of the systems and methods described herein, while “Manual 1,” “Manual 2,” and “Manual 3” all refer to uses of the previous system and method as shown in FIG. 2. The on-target rate that can be achieved with the present method can depend on the sample and its quality. In some cases, sequencing the eluted target molecule is achieved with an on-target base percentage of greater than about 50% when the sample is derived from preserved tissue, wherein the on-target base percentage is a proportion of the bases sequenced that are aligned to the target sequence. In some embodiments, sequencing the eluted target molecule is achieved with an on-target base percentage of greater than about 85% when the sample is derived from cfDNA, wherein the on-target base percentage is a proportion of the bases sequenced that are aligned to the target sequence. In some instances, sequencing the eluted target molecule is achieved with an on-target base percentage of greater than about 90% whenWSGR Reference No. 56252-721 .601 the sample is derived from genomic DNA, wherein the on-target base percentage is a proportion of the bases sequenced that are aligned to the target sequence.

[0115] FIG. 18 shows an example of on-target rates, comparing to the systems and methods described herein with a manual hybridization method. In this depicted example, “Present Method” refers to uses of the systems and methods described herein, while “Manual Method” refers to uses of the previous system and method as shown in FIG. 2. The present method performs slightly better covering 100% of the bases at > 20X for Exome and >100X for Pan Cancer.

[0116] FIG. 19 shows an example of sequencing coverage for a difficult to capture region, comparing to the systems and methods described herein with a manual hybridization method. In this depicted example, “Present Method” refers to uses of the systems and methods described herein, while “Manual Method” refers to uses of the previous system and method as shown in FIG. 2. Here, the EGFR gene was sequenced. A low frequency variant was found at the expected allele frequency.Table 2: Expected vs Observed frequency for an EGFR gene variant

[0117] In some cases, a plurality of target sequences are enriched. FIG. 20 shows an example of multiplexing, comparing to the systems and methods described herein with a manual hybridization method. The automated method described herein delivers consistently high on- target rate for high-plexity libraries using the Exome Panel. The automated method described herein delivers consistently high uniformity of coverage even when 20 samples are multiplexed. The automated method described herein enables cost reduction through high plexity, up to 20 samples.

[0118] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series ofWSGR Reference No. 56252-721 .601 numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0119] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0120] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WSGR Reference No. 56252-721 .601CLAIMSWHAT IS CLAIMED IS:

1. A method for enriching a nucleic acid molecule, the method comprising:(a) mixing (i) a nucleic acid sample comprising a target nucleic acid molecule having a target sequence with (ii) a probe having a nucleic acid sequence complimentary to the target sequence to hybridize the probe with the target nucleic acid molecule, wherein said mixing is performed at a mixing temperature that is within about 5% of a mixing set-point temperature;(b) binding the probe to a solid support while under continuous mixing, wherein said binding is performed at a binding temperature that is within about 5% of a binding set-point temperature;(c) washing the probe bound to the solid support to discard portions of the nucleic acid sample that are not hybridized with the probe, wherein said washing is performed at a washing temperature that is within about 5% of a washing set-point temperature; and(d) eluting the target nucleic acid molecule from the washed probe, thereby enriching the target nucleic acid molecule relative to the nucleic acid sample.

2. The method of claim 1, wherein (a)-(d) are performed on an electrowetting on dielectric (EWOD) device.

3. The method of claim 1 or 2, wherein (a) and (b) are performed concurrently.

4. The method of any one of claims 1 to 3, wherein (a)-(b) are completed in less than about 30 minutes.

5. The method of any one of claims 1 to 4, wherein (a)-(d) are completed in less than about 60 minutes.

6. The method of any one of claims 1 to 5, wherein the target molecule is eluted in a PCR master mix.

7. The method of claim 6, further comprising amplifying the eluted target molecule.

8. The method of claim 7, further comprising sequencing the eluted target molecule.

9. The method of any one of claims 1 to 8, further comprising sequencing the target molecule.

10. The method of any one of claims 1 to 9, wherein the probe is biotinylated.

11. The method of claim 10, wherein the probe is bound to a streptavidin solid support.

12. The method of any one of claims 1 to 11, wherein the solid support is magnetic.

13. The method of any one of claims 1 to 12, wherein the solid support is aggregated using a magnet.

14. The method of any one of claims 1 to 13, wherein:WSGR Reference No. 56252-721 .601(b) is commenced within 10 seconds of the conclusion of mixing; or(c) is commenced within 10 seconds of the conclusion of binding; or(d) is commenced within 10 seconds of the conclusion of washing; or any combination thereof.

15. The method of any one of claims 1 to 14, wherein the nucleic acid sample comprises a plurality of molecules having the target sequence.

16. The method of any one of claims 1 to 15, wherein a plurality of nucleic acid samples are processed in parallel with the nucleic acid sample on an EWOD device.

17. The method of any one of claims 1 to 16, wherein the solid support has a plurality of copies of the probe attached thereto.

18. The method of any one of claims 1 to 17, wherein mixing is performed in a droplet of fluid on a surface of an EWOD device in proximity to an array of electrodes.

19. The method of claim 18, wherein the droplet is subsequently transferred to a portion of the EWOD device not in proximity to the array of electrodes.

20. The method of any one of claims 1 to 19, wherein (a) forms a mixture of nucleic acid molecules comprising fragments of genomic DNA.

21. The method of claim 20, wherein the fragments are prepared by mechanical and / or enzymatic shearing.

22. The method of claim 20 or 21, wherein the fragments have adapters ligated thereto, wherein the adapters facilitate sequencing of the fragments.

23. The method of any one of claims 1 to 22, wherein hybridization is performed for at least about 4-22 hours.

24. The method of any one of claims 1 to 23, wherein hybridization is performed at a setpoint temperature of about 55 °C, about 65 °C, or about 75 °C.

25. The method of any one of claims 1 to 24, wherein any reagents that are added are prewarmed to a set-point temperature.

26. The method of any one of claims 1 to 25, wherein one or more consumables are utilized during (a)-(d), and wherein at least one of the one or more consumables is pre-warmed to a set-point temperature.

27. The method of claim 26, wherein the one or more consumables comprises a fluidic tip or beads.

28. The method of any one of claims 1 to 27, further comprising performing (c) a plurality of times.

29. The method of claim 28, wherein the bound probe is washed with a first wash buffer at 65°C.WSGR Reference No. 56252-721 .60130. The method of claim 29, further comprising washing the bound probe twice with a stringent wash buffer at 65 °C.

31. The method of claim 30, further comprising washing the bound probe with the first wash buffer at room temperature.

32. The method of any one of claims 29 to 31, further comprising washing the bound probe with a second wash buffer at room temperature.

33. The method of claim 32, further comprising washing the bound probe with a third wash buffer at room temperature.

34. The method of any one of claims 7 to 33, wherein sequencing the eluted target molecule is achieved with a fold-80 penalty of less than about 1.5, wherein the fold-80 penalty is an amount of additional sequencing coverage required to bring 80% of the sequenced bases up to an average coverage.

35. The method of any one of claims 7 to 34, wherein sequencing the eluted target molecule is achieved with an on-target base percentage of greater than about 50% when the sample is derived from preserved tissue, wherein the on-target base percentage is a proportion of the bases sequenced that are aligned to the target sequence.

36. The method of any one of claims 7 to 35, wherein sequencing the eluted target molecule is achieved with an on-target base percentage of greater than about 85% when the sample is derived from cfDNA, wherein the on-target base percentage is a proportion of the bases sequenced that are aligned to the target sequence.

37. The method of any one of claims 7 to 36, wherein sequencing the eluted target molecule is achieved with an on-target base percentage of greater than about 90% when the sample is derived from genomic DNA, wherein the on-target base percentage is a proportion of the bases sequenced that are aligned to the target sequence.

38. The method of any one of claims 1 to 37, wherein the target nucleic acid molecule is enriched at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, or at least about 1,000-fold relative to an initial concentration of the target nucleic acid molecule in the sample.

39. The method of any one of claims 1 to 38, wherein a plurality of target sequences is enriched using (a)-(d).

40. The method of claim 39, wherein the plurality of target sequences corresponds to an oncogene panel comprising the BRCA1, BRCA2, EGFR, and TP53 genes.

41. The method of claim 39, wherein the plurality of target sequences corresponds to a panel for detection of pathogens, characterizing drug metabolism, or characterizing patient therapeutic response.WSGR Reference No. 56252-721 .60142. The method of any one of claims 1-41, wherein the mixing set-point temperature, the binding set-point temperature, and the washing set-point temperature are each within about 5% of a common set-point temperature.

43. The method of claim 42, wherein the solid support, a hybridization buffer, a washing buffer, and / or an elution buffer are warmed to the common set-point temperature prior to being used in the method.

44. The method of any one of claims 1-43, wherein (b) is for at least 15 minutes.

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