Library-preparation-on-a-chip approach to metagenomic wastewater sequencing

Abstract

A library preparation device is a chip including (a) a biological sample well, (b) an end repair and elution well, downstream from the biological sample well, (c) an adapter ligation and elution well, downstream from the end repair and elution well, and (d) a final library well downstream from the ligation and elution well. The library preparation device is useful in a method of library preparation on a library preparation device.

Description

Docket No. 13177N-2957WOLIBRARY-PREPARATION-ON-A-CHIP APPROACH TO METAGENOMIC WASTEWATER SEQUENCINGRelated Application

[0001] This application claims the benefit of US Provisional Patent Application Serial No. 63 / 682,283 filed on August 12, 2024, the entirety of which is incorporated herein by reference.Statement Regarding Federally Sponsored Research or Development

[0002] This invention was made with government support under Grant Nos. 1U01DA053901- 01 and P30 ES026529 awarded by the National Institute of Health (NIH), Contract No. BAA 75D301-20-R-68024 awarded by the Centers for Disease Control and Prevention (CDC), and Grant Nos. 2154934 and 2412446 awarded by the National Science Foundation (NSF). The government has certain rights in the invention.Technical Field

[0003] This document relates generally to long-read library preparation for sequencing purposes and, more particularly, to an on-chip library preparation device and related method useful for metagenomic wastewater sequencing and other applications.Background

[0004] Sequencing-based diagnostic and public health surveillance is a growing field that aims to reduce the time-to-answer with unparalleled breadth in potential target analytes, such as drug resistance genes in wastewater treatment plants or single-nucleotide polymorphisms in liquid biopsies. However, most clinical and research sequencing cores only rely on short read Next-Gen Sequencing (NGS) which requires significant investment, expertise and computation power to perform. These requirements limit the use of this analysis endpoint to areas of the world with large, centralized laboratory infrastructure which may introduce significant shipping delays that lower the data actionability in a healthcare or public health setting.

[0005] This document relates to, a low-cost and simple-to-use library preparation device that will allow areas with minimal laboratory utilities (ImL pipette, hand-held or automated magnet, heat source) to generate a sequencing library that can be loaded into an Oxford Nanopore Technologies (ONT) sequencing device. This technology may be highly impactful in rural or low- resource settings where decentralized research groups or clinics are interested in pursuingDocket No. 13177N-2957WO sequencing, but do not have access to locally available sequencing services to perform library preparation. Our simplification and sealed chip approach to library preparation also allows for onsite or point-of-care testing by drastically reducing the chance for contamination and liquidhandling steps. Most groups choose to perform library preparation in a lateral flow hood or sterile laboratory due to the air-drying steps during more conventional solid-phase reversible immobilization (SPRI) wash steps.

[0006] Advantageously, we eliminate the need for air-drying by subbing conventional ethanol washes for a PEG / NaCL wash buffer loaded between oil wells to prevent leakage. Therefore, a user could extract nucleic acids using a variety of low-cost methods, and then immediately begin library preparations on our device. Our goal is to build a path towards immediate data collection to aid public health departments combating emerging pathogens, or clinicians who rely on diagnostic tools to better treat patients promptly.Summary

[0007] Each of the following terms written in singular grammatical form: “a”, “an”, and “the”, as used herein, means “at least one”, or “one or more”. Use of the phrase “One or more” herein does not alter this intended meaning of “a”, “an”, or “the”. Accordingly, the terms “a”, “an”, and “the”, as used herein, may also refer to, and encompass, a plurality of the stated entity or object, unless otherwise specifically defined or stated herein, or, unless the context clearly dictates otherwise. For example, the phrase: “a reagent”, as used herein, may also refer to, and encompass, a plurality of reagents.

[0008] Each of the following terms: “includes”, “including”, “has”, “having”, “comprises”, and “comprising”, and, their linguistic / grammatical variants, derivatives, or / and conjugates, as used herein, means “including, but not limited to”, and is to be taken as specifying the stated component(s), feature(s), characteristic(s), parameter(s), integer(s), or step(s), and does not preclude addition of one or more additional component(s), feature(s), characteristic(s), parameter(s), integer(s), step(s), or groups thereof.

[0009] The phrase “consisting of’, as used herein, is closed-ended and excludes any element, step, or ingredient not specifically mentioned. The phrase “consisting essentially of’, as usedDocket No. 13177N-2957WO herein, is a semi-closed term indicating that an item is limited to the components specified and those that do not materially affect the basic and novel characteristic(s) of what is specified.

[0010] Terms of approximation, such as the terms about, substantially, approximately, etc., as used herein, refers to ± 10 % of the stated numerical value.

[0011] In accordance with the purposes and benefits set forth herein, a library preparation device, comprises, consists of or consists essentially of a chip including:(a) a biological sample well adapted to receive extracted nucleic acids from a biological sample;(b) an end repair and elution well, downstream from the biological sample well, adapted to receive the extracted nucleic acids from the biological sample well and at least one end repair agent adapted to repair ends of the extracted nucleic acids and provide end-repaired nucleic acid fragments;(c) an adapter ligation and elution well, downstream from the end repair and elution well, adapted to receive the end-repaired nucleic acid fragments from the end repair and elution well and at least one adapter ligation agent adapted to join sequencing adapters to repaired ends of the extracted nucleic acid fragments; and(d) a final library well, downstream from the adapter ligation and elution well, adapted to receive the nucleic acid fragments joined with the sequencing adapters from the adapter ligation and elution well.

[0012] In at least some of the many possible embodiments, the device further includes (a) a first cleaning well group between the biological sample well and the end repair and elution well, (b) a second cleaning well group between the end repair and elution well and the adapter ligation and elution well, and (c) a third cleaning well group between the adapter ligation and elution well and the final library well. The first, second and third cleaning well groups may each comprise at least one wash well between an upstream isolation buffer well and a downstream isolation buffer well.

[0013] In some embodiments, the device further includes a first paramagnetic particle well in communication with the end repair and elution well. In some embodiments, the device further includes a first convoluted channel providing fluid communication between the first paramagneticDocket No. 13177N-2957WO particle well and the end repair and elution well. In some embodiments, the device further includes a first fluid medium well upstream from the first paramagnetic particle well.

[0014] In some embodiments, the device further includes a second paramagnetic particle well in communication with the adapter ligation and elution well. In some embodiments, the device further includes a second convoluted channel providing fluid communication between the second paramagnetic particle well and the adapter ligation and elution well. In some embodiments, the device further includes a second fluid medium well upstream from the second paramagnetic particle well.

[0015] In some embodiments, the device further includes a first plurality of paramagnetic particles bound with the extracted nucleic acids in the biological sample well. In some embodiments, the device further includes a second plurality of paramagnetic particles received in the first paramagnetic particle well and adapted to bind with the end-repaired nucleic acids in the end repair and elution well. In some embodiments, the device further includes a third plurality of paramagnetic particles received in the second paramagnetic particle well and adapted to bind with the nucleic acid fragments joined with the sequencing adapters in the adapter ligation and elution well.

[0016] In accordance with an additional aspect, a new and improved method of library preparation on a library preparation device, comprises, consists of or consists essentially of: (a) adding extracted nucleic acid fragments from a biological sample, (b) repairing ends of the extracted nucleic acid fragments, (c) joining sequencing adapters to repaired ends on the nucleic acid fragments, and (d) collecting a final library of nucleic acids with sequencing adapters wherein steps (a)-(d) are all completed on a single chip.

[0017] The method of library preparation on a library preparation device, may comprise, consist of or consist essentially of:(a) displacing extracted nucleic acid fragments from a biological sample well to an end repair and elution well;(b) repairing ends of the extracted nucleic acid fragments in the end repair and elution well to provide end-repaired nucleic acid fragments;Docket No. 13177N-2957WO(c) displacing the end-repaired nucleic acid fragments from the end repair and elution well to an adapter ligation and elution well;(d) joining sequencing adapters to repaired ends of the nucleic acid fragments in the adapter ligation and elution well; and(e) displacing the nucleic acid fragments joined with sequencing adapters to a final library well wherein the biological sample well, the end repair and elution well, the adapter ligation and elution well and the final library well are all in fluid communication on a single chip.

[0018] In some embodiments, the method further includes cleaning the extracted nucleic acid fragments in a first cleaning well group as the extracted nucleic acids are displaced from the biological sample well to the end repair and elution well. In some embodiments, the method further includes cleaning the end-repaired nucleic acid fragments in a second cleaning well group as the end-repaired nucleic acid fragments are displaced from the end repair and elution well to the ligation and elution well. In some embodiments, the method further includes cleaning the nucleic acid fragments joined with the sequencing adapters in a third cleaning well group as the nucleic acid fragments joined with the sequencing adapters are displaced from the adapter ligation and elution well to the final library well.

[0019] In some embodiments, the method further includes binding the extracted nucleic acid fragments to a first plurality of paramagnetic particles in the biological sample well. In some embodiments, the method further includes (a) releasing the extracted nucleic acid fragments from the first plurality of paramagnetic fragments and then (b) binding the end-repaired nucleic acid fragments to a second plurality of paramagnetic particles in the end repair and elution well.

[0020] In some embodiments, the method further includes (a) releasing the end-repaired nucleic acid fragments from the second plurality of paramagnetic particles and then (b) binding the nucleic acid fragments joined to the sequencing adapters to a third plurality of paramagnetic particles in the ligation and elution well. In some embodiments, the method further includes releasing the nucleic acid fragments joined to the sequencing adapters from the third plurality of paramagnetic particles in the library well. In some embodiments, the method further includes using a magnetic field to displace: (a) the first plurality of paramagnetic particles bound to the extractedDocket No. 13177N-2957WO nucleic acid fragments from the biological sample well, through the first cleaning well group to the end repair and elution well, (b) the second plurality of paramagnetic particles bound to the end- repaired nucleic acid fragments from the end repair and elution well, through the second cleaning well group to the adapter ligation and elution well, and (c) the third plurality of paramagnetic particles bound to the nucleic acid fragments joined to the sequencing adapters from the adapter ligation and elution well, through the third cleaning well group to the final library well.

[0021] In the following description, there are shown and described several different embodiments of (a) the library preparation device and (b) the related method of library preparation. As it should be realized, the device and the related methods are capable of other, different embodiments and their several details are capable of modification in various, obvious aspects all without departing from the system, instrument and method as set forth and described in the following claims. Accordingly, the drawings and descriptions should be regarded as illustrative in nature and not as restrictive.Brief Description of the Drawing Figures

[0022] The accompanying drawing figures incorporated herein and forming a part of the specification, illustrate certain aspects of the library preparation device and related method and together with the description serve to explain certain principles thereof. A person of ordinary skill in the art will readily recognize from the following discussion that alternative embodiments of the device and method may be employed without departing from the principles described below.

[0023] Figure 1 is a perspective view of the library preparation device in the form of a chip including a plurality of wells adapted for library preparation.

[0024] Figure 2 is a detailed schematic view showing how the nucleic acid fragments bound to the first plurality of paramagnetic particles are displaced from the biological sample well through the first microchannel to the first isolation well (Action Arrow A) and then through the second microchannel to the first wash well (Action Arrow B).Detailed Description

[0025] As shown in Figure 1, the library preparation device 10 may comprise a body, in the form of a chip 12, including a plurality of wells 14. The chip 12 may be fabricated fromDocket No. 13177N-2957WO substantially any appropriate material, including, for example, polymethyl methacrylate or acrylic, by laser cutting, 3D printing or other appropriate methods. In one particularly useful embodiment shown in Figure 1, the chip 12 includes a biological sample well 16, an end repair and elution well 18, an adapter ligation and elution well 20 and a final library well 22. All of these wells 16, 18, 20 and 22 are in fluid communication with each other as described in greater detail below.

[0026] A first cleaning well group 24 is provided between the biological sample well 16 and the end repair and elution well 18. In the illustrated embodiment, the first cleaning well group 24 comprises an upstream isolation buffer well 26, first and second cleaning wells 28, 30 and a downstream isolation buffer well 32. A second cleaning well group 34 is provided between the end repair and elution well 18 and the adapter ligation and elution well 20. In the illustrated embodiment, the second cleaning well group 34 comprises an upstream isolation buffer well 36, first and second cleaning wells 38, 40 and a downstream isolation buffer well 42. Similarly, a third cleaning well group 44 is provided between the adapter ligation and elution well 20 and the final library well 22. In the illustrated embodiment, the third cleaning well group 44 comprises an upstream isolation buffer well 46, first and second long fragment buffer wells 48, 50 and a downstream isolation buffer well 52.

[0027] A plurality of microchannels 54i-54is interconnect the wells 16, 26, 28, 30, 32, 18, 36, 38, 40, 42, 20, 46, 48, 50, 52 and 22 in series. As illustrated in Figure 1, the microchannels 54i- 5415 have a larger cross sectional area at the upstream end than the downstream end.

[0028] As also shown in Figure 1, the chip 12 further includes a first paramagnetic particle well 56 upstream from the end repair and elution well 18 and a first fluid media (e.g. oil) well 58 upstream from the first paramagnetic particle well. The flow channel 60 provides fluid communication between the wells 56, 58. A convoluted channel 62 provides fluid communication between the first paramagnetic particle well 56 and the end repair and elution well 18.

[0029] Similarly, the chip 12 further includes a second paramagnetic particle well 64 upstream from the adapter ligation and elution well 20 and a second fluid media (e.g. oil) well 66 upstream from the second paramagnetic particle well. The flow channel 68 provides fluid communication between the wells 64, 66. A convoluted channel 70 provides fluid communication between the second paramagnetic particle well 64 and the adapter ligation and elution well 20.Docket No. 13177N-2957WO

[0030] The biological sample well 16 is adapted to receive extracted nucleic acid fragments from a biological sample, such as a wastewater sample. The end repair and elution well 18, downstream from the biological sample well 16, is adapted to receive the extracted nucleic acid fragments from the biological sample well and at least one end repair agent, of a type known in the art, adapted to repair ends of the extracted nucleic acid fragments and provide end-repaired nucleic acid fragments.

[0031] The adapter ligation and elution well 20, downstream from the end repair and elution well 18, is adapted to receive the end-repaired nucleic acid fragments from the end repair and elution well and at least one adapter ligation agent, of a type known in the art, adapted to join sequencing adapters to the repaired ends of the extracted nucleic acid fragment. The final library well 22, downstream from the adapter ligation and elution well 20, is adapted to receive the nucleic acid fragments joined with the sequencing adapters from the adapter ligation and elution well.

[0032] In use, if the wells 14 in the chip 12 are not presealed at the bottom, they may be sealed with a strip of PCR tape. The isolation buffer wells 26, 32, 36, 42, 46, 52 and the two liquid media wells 58, 66 may then be filled with one to two drops of a silicone oil having a viscosity of perhaps 1000 cSt pl of bead(l :2 mixing ration of 50 cSt and 10000 cSt silicone oils available from Sigma Aldrich, 378356 and 378402). The wash wells 28, 30, 38, 40, are filled with about 150pl of a DNA wash, such as, 250pl 5M sodium chloride (NaCl) solution, 250pl 50% (w / v) polyethylene glycol (PEG) 8000, and 500pl of DNA solution. The DNA solution may comprise 45mL nuclease- free water, 500pl 1 M tri-base, 500pl 0.1 M disodium EDTA, 250pl 10% Tween 20 with enough 1 N hydrochloric acid (HC1) added to bring the pH to 8.0. The final volume may then be brought up to 50mL using nuclease-free water.

[0033] Approximately 150pl of long fragment buffer is added to each of the long fragment buffer wells 48, 50. As is known in the art, long fragment buffer contains reagents necessary for the amplification and sequencing of DNA fragments, typically having a length of 3kb or longer. Approximately 60pl of paramagnetic particles or beads P2 are added to the first paramagnetic particle well 56. Similarly, approximately 60 pL of paramagnetic particles or beads P3 are added to the second paramagnetic particle well 64.Docket No. 13177N-2957WO

[0034] Approximately 60pl of end repair solution, adapted to repair ends of the nucleic acid fragments, is added to the end repair and elution well 18. Such a solution may comprise 48pl of nuclease-free water, 7pl of NEBNext FFPE DNA Repair Buffer v2, 2pl of NEBNext FFPE DNA Repair Mix and 3 pl of Ultra II End-Prep Enzyme Mix.

[0035] Approximately 60 pl of nuclease-free water is added to the adapter ligation and elution well 20 and approximately 15 pl of nuclease-free water is added to the final library well 22.

[0036] In one possible embodiment, the nucleic acid fragments are extracted from the biological sample / wastewater sample by first mixing the sample, with a lysis buffer and a first plurality of paramagnetic particles or beads Pl in a vortex mixer in accordance with procedures known in the art. The lysis buffer is adapted to break open and release the contents of the biological cells in the sample. In one possible embodiment, the lysis buffer comprises a mixture of guanidine thiocyanate and 4-morpholine-thanesulfonic acid sodium salt in a solvent of ethanol and deionized water. The lysis buffer also includes an ionic surfactant such as Tween 20. The plurality of paramagnetic particles are also of a type known in the art as solid-phase reversible immobilization (SPRI) beads, such as available in Ligation Sequencing Kit V14 (SQK-LSK114). These particles / beads Pl may be silica-coated and are adapted to bind to the nucleic acid fragments of a biological pathogen.

[0037] The first plurality of paramagnetic particles Pl bound to the extracted nucleic acid fragments are then mixed with nuclease free water and added in an appropriate amount (approximately 400pl) to the biological sample well 16. The wells 14 are then sealed at the top by of the chip 12 with PCR tape. As shown in Figure 2, a magnet M may be placed near or against the bottom of the chip 12 below the biological sample well 16. The magnet M generates a magnetic field that may be used to pellet the particles Pl together. The elongated, narrow shape of the well 16 helps make this task easier. As best shown in Figure 2, the magnet M is then slowly moved, at a pace of about 2-5 mm per second, in the direction of Action Arrow A so that the paramagnetic particles Pl bound to the nucleic acid fragments F, following the magnetic field, pass through the first microchannel 54i into the upstream isolation well 26 and then through the second microchannel 542 to the first wash well 28 (note Action Arrow B).Docket No. 13177N-2957WO

[0038] The immiscibility of the fluid in the sample well 16 with the oil in the isolation buffer well 26 serves as a barrier so that the fluid in the sample well does not pass through the microchannel 54i into the isolation well 26. Thus, the particles Pl are cleaned of water, lysis buffer and other residues. Similarly, the immiscibility of the oil in the upstream isolation buffer well 26 with the DNA wash in the wash well 28 acts as a barrier so that the oil in the upstream isolation well does not pass through the microchannel 542 into the wash well 28.

[0039] The paramagnetic particles Pl are then allowed to sit in the wash well 28 for about one minute before once again being moved by magnet M through the next microchannel 54s to the second wash well 30 where the particles are again allowed to sit for about one minute. At that point, the magnet M is once again used to move or displace the particles Pl through the microchannel 44 to the downstream isolation buffer well 32 where the immiscibility of the DNA wash in the wash well 30 with the oil in the downstream isolation well 32 once again acts as a barrier to passage. The paramagnetic particles Pl continue to be moved under the force of the magnetic field of the magnet M through the microchannel 54s to the end repair and elution well 18.

[0040] At this point, the magnet M is removed from the underside of the chip 12 and an electromagnet is placed over the end repair and elution well 18. The wires of the electromagnet are attached to the normally open side of a relay switch and for the first two minutes, the electromagnet is operated for 3 seconds on and 40 seconds off. For the next 3.5 minutes, the electromagnet is operated for 0.5 seconds on and 10 seconds off. For the next 4.5 minutes, the electromagnet is operated for 0.5 seconds on and 15 seconds off. These times are approximate and should be considered as representative of one possible embodiment of the method and not as exclusive. This mixing action provided by the electromagnet functions to elude or release the extracted nucleic acid fragments from the particles Pl.

[0041] The paramagnetic particles Pl are then repelled from the electromagnet and the electromagnet is removed. The magnet M is then applied to the underside of the well 18 to collect the particles Pl in the magnetic field and then shifted to the left to move those particles back upstream through the microchannel 54s into the isolation well 32. Once again, the oil in the well 32 acts as a barrier to prevent the passage of the immiscible fluid in the well 18 from passingDocket No. 13177N-2957WO through the microchannel 54s to the well 32. Thus, the extracted nucleic acid fragments, now with repaired ends, are maintained in the well 18.

[0042] The end repair and elution well is then heated at about 20° C for about 5 minutes and then to about 65° C for about 5 minutes. This heating of the reagents and extracted nucleic fragments in the well 18 promotes the repair to the ends of the nucleic acid fragments.

[0043] Next, a lOOOmL pipette or other instrument is inserted into the small well 57 upstream of the oil well 58. By depressing the pipette, pressure is applied to push the oil from the well 58 into the first paramagnetic particle well, which in turn, pushes the particles / beads P2though the first convoluted channel 62 into the end repair and elution well 18. The electromagnet may then be used with the same protocol described above to mix the particles / beads P2 with the end-repaired nucleic acid fragments in the end repair and elution well 18 in order to promote binding of the end- repaired nucleic acid fragments with the particles / beads P2.

[0044] After removing the electromagnet, the magnet M is applied to the underside of the chip at the well 18 to form the particles / beads P2 into a pellet. That pellet of particles / beads P2 is then moved with the magnet M by the force of its magnetic field through the second cleaning well group 34 (i.e. serially through the wells 36, 38, 40 and 42 via the microchannels 54e-54io) into the adapter ligation and elution well 20. As this is done, the reagents in the wash wells 38, 40 clean the nucleic acid fragments bound to the particles / beads P2 while the immiscibility barrier established by the oil in the isolation wells 36 and 42 prevents the passage of fluid between the wells in the manner described above for the first cleaning well group 24.

[0045] At this point, the magnet M is removed from the underside of the chip 12 and an electromagnet is placed over the adapter ligation and elution well 20. The wires of the electromagnet are attached to the normally open side of a relay switch and for the first two minutes, the electromagnet is operated for 3 seconds on and 40 seconds off. For the next 3.5 minutes, the electromagnet is operated for 0.5 seconds on and 10 seconds off. For the next 4.5 minutes, the electromagnet is operated for 0.5 seconds on and 15 seconds off. These times are approximate and should be considered as representative of one possible embodiment of the method and not as exclusive. This mixing action provided by the electromagnet functions to elute or release the end- repaired nucleic acid fragments from the particles / beads P2.Docket No. 13177N-2957WO

[0046] About 40pl of adapter ligation solution, adapted to join sequencing adapters to the repaired ends of the extracted nucleic acid fragments, is then added to the well 20 and the chip is then incubated at room temperature for about 10 minutes. This is done by using a pipette to punch holes in the PCRtape with the solution addition made in the small wells 72 in communication with the well 20. The adapter ligation solution may comprise 5 pl of Ligation Adapter (LA), 25 l of Ligation Buffer (LNB) and lOpl Salt-T4® DNA Ligase.

[0047] Next, a lOOOmL pipette or other instrument is inserted into the small well 65 upstream of the oil well 66. By depressing the pipette, pressure is applied to push the oil from the well 66 into the second paramagnetic particle well 64, which in turn, pushes the particles / beads P3 though the second convoluted channel 70 into the adapter ligation and elution well 20. The electromagnet may then be used with the same protocol described above to mix the parti cl es / beads P3 with the nucleic acid fragments j oined with sequencing adapters in the adapter ligation and elution well 20 in order to promote binding of the nucleic acid fragments joined with sequencing adapters with the parti cles / beads P3.

[0048] After removing the electromagnet, the magnet M is applied to the underside of the chip at the well 20 to form the particles / beads P3 into a pellet. That pellet of particles / beads P3 is then moved with the magnet M, by the force of its magnetic field, through the third cleaning well group 44 (i.e. serially through the wells 46, 48, 50 and 52 via the microchannels 54n-54is) into the final library well 22. As this is done, the long fragment buffer in the wash wells 48, 50 functions to maintain the integrity and fidelity of the nucleic acid fragments joined with the sequencing adapters that are bound to the particles / beads. The immiscibility barrier established by the oil in the isolation wells 46 and 52 prevents the passage of fluid between the wells in the manner described above for the first cleaning well groups 24 and 34.

[0049] Once the particles / beads P3 are in the final library well 22, the nucleic acid fragments joined with the sequencing adapters are eluded from the particles / beads P3 by mixing with a pipet or other means. The contents of the well 24 are then allowed to sit for about five minutes. Afterward, the magnet M is used to move the particles / beads from the final library well 24, through the microchannel 5415, back upstream to the isolation well 52. As this is done, the oil in the wellDocket No. 13177N-2957WO52 acts as a barrier to maintain the now free nucleic acid fragments joined with the sequencing adapters in the well 24.

[0050] The library of nucleic acid fragments joined with the sequencing adapters is then ready to be loaded following Oxford Nanopore Technologies (ONT) instructions.

[0051] Consistent with the above description, a method of library preparation on a library preparation device includes the steps of:(a) adding extracted nucleic acid fragments from a biological sample;(b) repairing ends of the extracted nucleic acid fragments;(c) joining sequencing adapters to repaired ends on the nucleic acid fragments; and(d) collecting a final library of nucleic acid with sequencing adapters wherein (a), (b), (c) and (d) are all completed on a single chip 12.

[0052] More specifically, that method of library preparation on a library preparation device 10, includes:(a) displacing extracted nucleic acid fragments from a biological sample well 16 to an end repair and elution well 18;(b) cleaning the extracted nucleic acid fragments in a first cleaning well group 24 as the extracted nucleic acids are displaced from the biological sample well 16 to the end repair and elution well 18;(c) (i)releasing the extracted nucleic acid fragments from a first plurality of paramagnetic particles Pl, (ii) repairing ends of the extracted nucleic acid fragments in the end repair and elution well 18 to provide end-repaired nucleic acid fragments, and (iii) binding the end-repaired nucleic acid fragments to a second plurality of paramagnetic particles P2 in the end repair and elution well,(d) displacing the end-repaired nucleic acid fragments from the end repair and elution well 18 to an adapter ligation and elution well 20;(e) cleaning the end-repaired nucleic acid fragments in a second cleaning well group 34 as the end-repaired nucleic acid fragments are displaced from the end repair and elution well 18 to the adapter ligation and elution well 20;Docket No. 13177N-2957WO(f) (i) releasing the end-repaired nucleic acid fragments from the second plurality of paramagnetic particles P2, (ii) joining sequencing adapters to repaired ends of the nucleic acid fragments in the adapter ligation and elution well 20, and (iii) binding the nucleic acid fragments joined with sequencing adapters to a third plurality of paramagnetic particles P3 in the ligation and elution well; and(g) and displacing the nucleic acid fragments joined with sequencing adapters to a final library well 22.

[0053] The biological sample well 16, the end repair and elution well 18, the ligation and elution well 20 and the final library well 22 are all in fluid communication on a single chip 12. Steps (a)-(g) are accomplished by using a magnetic field to displace: (a) the first plurality of paramagnetic particles bound to the extracted nucleic acid fragments from the biological sample well, through the first cleaning well group to the end repair and elution well, (b) the second plurality of paramagnetic particles bound to the end-repaired nucleic acid fragments from the end repair and elution well, through the second cleaning well group to the ligation and elution well, and (c) the third plurality of paramagnetic particles bound to the nucleic acid fragments joined with sequencing adapters from the ligation and elution well, through the third cleaning well group to the final library well.

[0054] It may be said that this document relates to the following items.1. A library preparation device, comprising: a chip including(a) a biological sample well adapted to receive extracted nucleic acid fragments from a biological sample;(b) an end repair and elution well downstream from the biological sample well, said end repair and elution well adapted to receive the extracted nucleic acid fragments from the biological sample well and at least one end repair agent adapted to repair ends of the extracted nucleic acid fragments and provide end-repaired nucleic acid fragments;(c) an adapter ligation and elution well downstream from the end repair and elution well, said adapter ligation and elution well adapted to receive the end-repaired nucleic acid fragments from the end repair and elution well and at least one adapter ligation agent adapted to join sequencing adapters to repaired ends of the extracted nucleic acid fragments; andDocket No. 13177N-2957WO(d) a final library well downstream from the ligation and elution well adapted to receive the nucleic acid fragments joined with sequencing adapters from the adapter ligation and elution well.2. The library preparation device of item 1, further including (a) a first cleaning well group between the biological sample well and the end repair and elution well, (b) a second cleaning well group between the end repair and elution well and the ligation and elution well, and (c) a third cleaning well group between the ligation and elution well and the final library well.3. The library preparation device of item 2, wherein the first cleaning well group includes at least one wash well between an upstream isolation buffer well and a downstream isolation buffer well.4. The library preparation device of item 2, wherein the second cleaning well group includes at least one wash well between an upstream isolation buffer well and a downstream isolation buffer well.5. The library preparation device of item 2, wherein the third cleaning well group includes at least one long fragment buffer well between an upstream isolation buffer well and a downstream isolation buffer well.6. The library preparation device of item 2, further including a first paramagnetic particle well in communication with the end repair and elution well.7. The library preparation device of item 6, further including a first convoluted channel providing fluid communication between the first paramagnetic particle well and the end repair and elution well.8. The library preparation device of item 7, further including a first fluid medium well upstream from the first paramagnetic particle well.9. The library preparation device of item 8, further including a second paramagnetic particle well in communication with the ligation and elution well.Docket No. 13177N-2957WO10. The library preparation device of item 9, further including a second convoluted channel providing fluid communication between the second paramagnetic particle well and the ligation and elution well.11. The library preparation device of item 7, further including a second fluid medium well upstream from the second paramagnetic particle well.12. The library preparation device of item 11, further including a first plurality of paramagnetic particles bound with the extracted nucleic acid fragments in the biological sample well.13. The library preparation device of item 12, further including a second plurality of paramagnetic particles received in the first paramagnetic particle well and adapted to bind with the end-repaired nucleic acid fragments in the end repair and elution well.14. The library preparation device of item 13, further including a third plurality of paramagnetic particles received in the second paramagnetic particle well and adapted to bind with the nucleic acid fragments joined with sequencing adapters in the adapter ligation and elution well.15. A method of library preparation on a library preparation device, comprising:(a) adding extracted nucleic acid fragments from a biological sample;(b) repairing ends of the extracted nucleic acid fragments;(c) joining sequencing adapters to repaired ends on the nucleic acid fragments; and(d) collecting a final library of nucleic acids with sequencing adapters wherein (a), (b), (c) and (d) are all completed on a single chip.16. A method of library preparation on a library preparation device, comprising: displacing extracted nucleic acid fragments from a biological sample well to an end repair and elution well; repairing ends of the extracted nucleic acid fragments in the end repair and elution well to provide end-repaired nucleic acid fragments; displacing the end-repaired nucleic acid fragments from the end repair and elution well to an adapter ligation and elution well;Docket No. 13177N-2957WO joining sequencing adapters to repaired ends of the nucleic acid fragments in the adapter ligation and elution well; and displacing the nucleic acid fragments joined with sequencing adapters to a final library well wherein the biological sample well, the end repair and elution well, the adapter ligation and elution well and the final library well are all in fluid communication on a single chip.17. The method of item 16, further including cleaning the extracted nucleic acid fragments in a first cleaning well group as the extracted nucleic acids are displaced from the biological sample well to the end repair and elution well.18. The method of item 17, further including cleaning the end-repaired nucleic acid fragments in a second cleaning well group as the end-repaired nucleic acid fragments are displaced from the end repair and elution well to the adapter ligation and elution well.19. The method of item 18, further including cleaning the nucleic acid fragments j oined with sequencing adapters in a third cleaning well group as the nucleic acid fragments joined with sequencing adapters are displaced from the adapter ligation and elution well to the final library well.20. The method of item 19, further including (a) releasing the extracted nucleic acid fragments from the first plurality of paramagnetic particles and then (b) binding the end-repaired nucleic acid fragments to a second plurality of paramagnetic particles in the end repair and elution well.21. The method of item 20, further including (a) releasing the end-repaired nucleic acid fragments from the second plurality of paramagnetic particles and then (b) binding the nucleic acid fragments joined with sequencing adapters to a third plurality of paramagnetic particles in the ligation and elution well.22. The method of item 21, further including releasing the nucleic acid fragments joined with sequencing adapters from the third plurality of paramagnetic particles in the library well.23. The method of item 22, further including using a magnetic field to displace: (a) the first plurality of paramagnetic particles bound to the extracted nucleic acid fragments from theDocket No. 13177N-2957WO biological sample well, through the first cleaning well group to the end repair and elution well, (b) the second plurality of paramagnetic particles bound to the end-repaired nucleic acid fragments from the end repair and elution well, through the second cleaning well group to the ligation and elution well, and (c) the third plurality of paramagnetic particles bound to the nucleic acid fragments joined with sequencing adapters from the ligation and elution well, through the third cleaning well group to the final library well.

[0055] Although the library preparation device and the related method of library preparation of this disclosure have been illustratively described and presented by way of specific exemplary embodiments, and examples thereof, it is evident that many alternatives, modifications, or / and variations, thereof, will be apparent to those skilled in the art. Accordingly, it is intended that all such alternatives, modifications, or / and variations, fall within the spirit of, and are encompassed by, the broad scope of the appended claims.

Claims

Docket No. 13177N-2957WOWhat is claimed;1. A library preparation device, comprising: a chip including(a) a biological sample well adapted to receive extracted nucleic acid fragments from a biological sample;(b) an end repair and elution well downstream from the biological sample well, said end repair and elution well adapted to receive the extracted nucleic acid fragments from the biological sample well and at least one end repair agent adapted to repair ends of the extracted nucleic acid fragments and provide end-repaired nucleic acid fragments;(c) an adapter ligation and elution well downstream from the end repair and elution well, said adapter ligation and elution well adapted to receive the end-repaired nucleic acid fragments from the end repair and elution well and at least one adapter ligation agent adapted to join sequencing adapters to repaired ends of the extracted nucleic acid fragments; and(d) a final library well downstream from the ligation and elution well adapted to receive the nucleic acid fragments joined with sequencing adapters from the adapter ligation and elution well.

2. The library preparation device of claim 1, further including (a) a first cleaning well group between the biological sample well and the end repair and elution well, (b) a second cleaning well group between the end repair and elution well and the ligation and elution well, and (c) a third cleaning well group between the ligation and elution well and the final library well.

3. The library preparation device of claim 2, wherein the first cleaning well group includes at least one wash well between an upstream isolation buffer well and a downstream isolation buffer well.

4. The library preparation device of claim 2, wherein the second cleaning well group includes at least one wash well between an upstream isolation buffer well and a downstream isolation buffer well.

5. The library preparation device of claim 2, wherein the third cleaning well group includes at least one long fragment buffer well between an upstream isolation buffer well and a downstream isolation buffer well.Docket No. 13177N-2957WO6. The library preparation device of claim 2, further including a first paramagnetic particle well in communication with the end repair and elution well.

7. The library preparation device of claim 6, further including a first convoluted channel providing fluid communication between the first paramagnetic particle well and the end repair and elution well.

8. The library preparation device of claim 7, further including a first fluid medium well upstream from the first paramagnetic particle well.

9. The library preparation device of claim 8, further including a second paramagnetic particle well in communication with the ligation and elution well.

10. The library preparation device of claim 9, further including a second convoluted channel providing fluid communication between the second paramagnetic particle well and the ligation and elution well.

11. The library preparation device of claim 7, further including a second fluid medium well upstream from the second paramagnetic particle well.

12. The library preparation device of claim 11, further including a first plurality of paramagnetic particles bound with the extracted nucleic acid fragments in the biological sample well.

13. The library preparation device of claim 12, further including a second plurality of paramagnetic particles received in the first paramagnetic particle well and adapted to bind with the end-repaired nucleic acid fragments in the end repair and elution well.

14. The library preparation device of claim 13, further including a third plurality of paramagnetic particles received in the second paramagnetic particle well and adapted to bind with the nucleic acid fragments joined with sequencing adapters in the adapter ligation and elution well.

15. A method of library preparation on a library preparation device, comprising:(a) adding extracted nucleic acid fragments from a biological sample;Docket No. 13177N-2957WO(b) repairing ends of the extracted nucleic acid fragments;(c) joining sequencing adapters to repaired ends on the nucleic acid fragments; and(d) collecting a final library of nucleic acids with sequencing adapters wherein (a), (b), (c) and (d) are all completed on a single chip.

16. A method of library preparation on a library preparation device, comprising: displacing extracted nucleic acid fragments from a biological sample well to an end repair and elution well; repairing ends of the extracted nucleic acid fragments in the end repair and elution well to provide end-repaired nucleic acid fragments; displacing the end-repaired nucleic acid fragments from the end repair and elution well to an adapter ligation and elution well; joining sequencing adapters to repaired ends of the nucleic acid fragments in the adapter ligation and elution well; and displacing the nucleic acid fragments joined with sequencing adapters to a final library well wherein the biological sample well, the end repair and elution well, the adapter ligation and elution well and the final library well are all in fluid communication on a single chip.

17. The method of claim 16, further including cleaning the extracted nucleic acid fragments in a first cleaning well group as the extracted nucleic acids are displaced from the biological sample well to the end repair and elution well.

18. The method of claim 17, further including cleaning the end-repaired nucleic acid fragments in a second cleaning well group as the end-repaired nucleic acid fragments are displaced from the end repair and elution well to the adapter ligation and elution well.

19. The method of claim 18, further including cleaning the nucleic acid fragments j oined with sequencing adapters in a third cleaning well group as the nucleic acid fragments joined with sequencing adapters are displaced from the adapter ligation and elution well to the final library well.

20. The method of claim 19, further including (a) releasing the extracted nucleic acid fragments from the first plurality of paramagnetic particles and then (b) binding the end-repairedDocket No. 13177N-2957WO nucleic acid fragments to a second plurality of paramagnetic particles in the end repair and elution well.

21. The method of claim 20, further including (a) releasing the end-repaired nucleic acid fragments from the second plurality of paramagnetic particles and then (b) binding the nucleic acid fragments joined with sequencing adapters to a third plurality of paramagnetic particles in the ligation and elution well.

22. The method of claim 21, further including releasing the nucleic acid fragments joined with sequencing adapters from the third plurality of paramagnetic particles in the library well.

23. The method of claim 22, further including using a magnetic field to displace: (a) the first plurality of paramagnetic particles bound to the extracted nucleic acid fragments from the biological sample well, through the first cleaning well group to the end repair and elution well, (b) the second plurality of paramagnetic particles bound to the end-repaired nucleic acid fragments from the end repair and elution well, through the second cleaning well group to the ligation and elution well, and (c) the third plurality of paramagnetic particles bound to the nucleic acid fragments joined with sequencing adapters from the ligation and elution well, through the third cleaning well group to the final library well.

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