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A microfluidic device for preparing next generation DNA sequencing libraries and for automating other laboratory protocols that require one or more column chromatography steps.

Tan SJ, Phan H, Gerry BM, Kuhn A, Hong LZ, Min Ong Y, Poon PS, Unger MA, Jones RC, Quake SR, Burkholder WF - PLoS ONE (2013)

Bottom Line: Library preparation for next-generation DNA sequencing (NGS) remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization.Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation.The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Proteos, Singapore.

ABSTRACT
Library preparation for next-generation DNA sequencing (NGS) remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization. We describe a microfluidic device for automating laboratory protocols that require one or more column chromatography steps and demonstrate its utility for preparing Next Generation sequencing libraries for the Illumina and Ion Torrent platforms. Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation. Using an appropriate column matrix and buffers, size selection can be performed on-chip following end-repair, dA tailing, and linker ligation, so that the libraries eluted from the chip are ready for sequencing. The core architecture of the device ensures uniform, reproducible column packing without user supervision and accommodates multiple routine protocol steps in any sequence, such as reagent mixing and incubation; column packing, loading, washing, elution, and regeneration; capture of eluted material for use as a substrate in a later step of the protocol; and removal of one column matrix so that two or more column matrices with different functional properties can be used in the same protocol. The microfluidic device is mounted on a plastic carrier so that reagents and products can be aliquoted and recovered using standard pipettors and liquid handling robots. The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors. In addition to NGS library preparation, the device and controller are suitable for automating other time-consuming and error-prone laboratory protocols requiring column chromatography steps, such as chromatin immunoprecipitation.

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Microfluidic device for automated NGS library preparation.(a) Automated multi-column device mounted on a plastic carrier that provides wells for loading samples and reagents and for pressurized operation of the device. The wells used to load reagents for NGS library preparation are labeled. Chromatography columns for the selective binding and release of DNA were formed either with ChargeSwitch beads or with carboxylated beads. Reagents that were used exclusively with the carboxylated beads are labeled in green. (b) Schematic of single reactor unit for reaction mixing and DNA purification. The regions denoted in the reaction circuit are as follows: Green, Sample; Orange, Buffer; Blue, Enzyme. Red solid rectangular boxes represent activated valves that partition the individual circuits. (c) Parallelization of 16 reactors on chip for preparation of up to 16 independent libraries. Layout of the entire device without the valve map showing reagent inlets and the design for multiplex library generation. The serpentine metering channel designed to ensure reliable column packing is highlighted in orange. (d) Schematics showing cross-sections of purification columns loaded with either 1) ChargeSwitch beads, which are held in place with a frit layer and a cap layer formed by larger beads, or 2) carboxylated beads.
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pone-0064084-g001: Microfluidic device for automated NGS library preparation.(a) Automated multi-column device mounted on a plastic carrier that provides wells for loading samples and reagents and for pressurized operation of the device. The wells used to load reagents for NGS library preparation are labeled. Chromatography columns for the selective binding and release of DNA were formed either with ChargeSwitch beads or with carboxylated beads. Reagents that were used exclusively with the carboxylated beads are labeled in green. (b) Schematic of single reactor unit for reaction mixing and DNA purification. The regions denoted in the reaction circuit are as follows: Green, Sample; Orange, Buffer; Blue, Enzyme. Red solid rectangular boxes represent activated valves that partition the individual circuits. (c) Parallelization of 16 reactors on chip for preparation of up to 16 independent libraries. Layout of the entire device without the valve map showing reagent inlets and the design for multiplex library generation. The serpentine metering channel designed to ensure reliable column packing is highlighted in orange. (d) Schematics showing cross-sections of purification columns loaded with either 1) ChargeSwitch beads, which are held in place with a frit layer and a cap layer formed by larger beads, or 2) carboxylated beads.

Mentions: The AMCC chip was designed to enable the automation of multistep laboratory protocols that combine one or more column chromatography steps with steps in which reagents are mixed and incubated. The chip is a prototype integrated fluidic circuit (IFC) developed and provided by Fluidigm Corp, South San Francisco. It was made using multi-layer soft lithography (MSL) methods [19] and contains independent modules for processing and recovering 16 different samples in parallel. For ease of use, the chip is mounted on a plastic carrier that contains wells for loading samples and reagents and for recovering processed samples (Figure 1A). The size of the carrier and the spacing between wells are the same as a standard 384 well plate, conforming to the Society for Biomolecular Screening (SBS) microplate format [23] and is thus compatible with standard multichannel pipettors and robotic workstations. In the current design, the chip provides two wells for the bead slurry used to form the column matrix, six wells for reagents used in the protocol (labeled "Enzymes" and "Reaction buffers" in Figure 1A), and wells for the binding, wash, and elution buffers used in the column chromatography steps, including four large-volume wells of 175 µl each (Figure 1A). In addition, 36 wells connect to the control lines used to actuate the microfluidic valves on the device, and two wells are used to purge channels with air.


A microfluidic device for preparing next generation DNA sequencing libraries and for automating other laboratory protocols that require one or more column chromatography steps.

Tan SJ, Phan H, Gerry BM, Kuhn A, Hong LZ, Min Ong Y, Poon PS, Unger MA, Jones RC, Quake SR, Burkholder WF - PLoS ONE (2013)

Microfluidic device for automated NGS library preparation.(a) Automated multi-column device mounted on a plastic carrier that provides wells for loading samples and reagents and for pressurized operation of the device. The wells used to load reagents for NGS library preparation are labeled. Chromatography columns for the selective binding and release of DNA were formed either with ChargeSwitch beads or with carboxylated beads. Reagents that were used exclusively with the carboxylated beads are labeled in green. (b) Schematic of single reactor unit for reaction mixing and DNA purification. The regions denoted in the reaction circuit are as follows: Green, Sample; Orange, Buffer; Blue, Enzyme. Red solid rectangular boxes represent activated valves that partition the individual circuits. (c) Parallelization of 16 reactors on chip for preparation of up to 16 independent libraries. Layout of the entire device without the valve map showing reagent inlets and the design for multiplex library generation. The serpentine metering channel designed to ensure reliable column packing is highlighted in orange. (d) Schematics showing cross-sections of purification columns loaded with either 1) ChargeSwitch beads, which are held in place with a frit layer and a cap layer formed by larger beads, or 2) carboxylated beads.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3722208&req=5

pone-0064084-g001: Microfluidic device for automated NGS library preparation.(a) Automated multi-column device mounted on a plastic carrier that provides wells for loading samples and reagents and for pressurized operation of the device. The wells used to load reagents for NGS library preparation are labeled. Chromatography columns for the selective binding and release of DNA were formed either with ChargeSwitch beads or with carboxylated beads. Reagents that were used exclusively with the carboxylated beads are labeled in green. (b) Schematic of single reactor unit for reaction mixing and DNA purification. The regions denoted in the reaction circuit are as follows: Green, Sample; Orange, Buffer; Blue, Enzyme. Red solid rectangular boxes represent activated valves that partition the individual circuits. (c) Parallelization of 16 reactors on chip for preparation of up to 16 independent libraries. Layout of the entire device without the valve map showing reagent inlets and the design for multiplex library generation. The serpentine metering channel designed to ensure reliable column packing is highlighted in orange. (d) Schematics showing cross-sections of purification columns loaded with either 1) ChargeSwitch beads, which are held in place with a frit layer and a cap layer formed by larger beads, or 2) carboxylated beads.
Mentions: The AMCC chip was designed to enable the automation of multistep laboratory protocols that combine one or more column chromatography steps with steps in which reagents are mixed and incubated. The chip is a prototype integrated fluidic circuit (IFC) developed and provided by Fluidigm Corp, South San Francisco. It was made using multi-layer soft lithography (MSL) methods [19] and contains independent modules for processing and recovering 16 different samples in parallel. For ease of use, the chip is mounted on a plastic carrier that contains wells for loading samples and reagents and for recovering processed samples (Figure 1A). The size of the carrier and the spacing between wells are the same as a standard 384 well plate, conforming to the Society for Biomolecular Screening (SBS) microplate format [23] and is thus compatible with standard multichannel pipettors and robotic workstations. In the current design, the chip provides two wells for the bead slurry used to form the column matrix, six wells for reagents used in the protocol (labeled "Enzymes" and "Reaction buffers" in Figure 1A), and wells for the binding, wash, and elution buffers used in the column chromatography steps, including four large-volume wells of 175 µl each (Figure 1A). In addition, 36 wells connect to the control lines used to actuate the microfluidic valves on the device, and two wells are used to purge channels with air.

Bottom Line: Library preparation for next-generation DNA sequencing (NGS) remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization.Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation.The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Proteos, Singapore.

ABSTRACT
Library preparation for next-generation DNA sequencing (NGS) remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization. We describe a microfluidic device for automating laboratory protocols that require one or more column chromatography steps and demonstrate its utility for preparing Next Generation sequencing libraries for the Illumina and Ion Torrent platforms. Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation. Using an appropriate column matrix and buffers, size selection can be performed on-chip following end-repair, dA tailing, and linker ligation, so that the libraries eluted from the chip are ready for sequencing. The core architecture of the device ensures uniform, reproducible column packing without user supervision and accommodates multiple routine protocol steps in any sequence, such as reagent mixing and incubation; column packing, loading, washing, elution, and regeneration; capture of eluted material for use as a substrate in a later step of the protocol; and removal of one column matrix so that two or more column matrices with different functional properties can be used in the same protocol. The microfluidic device is mounted on a plastic carrier so that reagents and products can be aliquoted and recovered using standard pipettors and liquid handling robots. The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors. In addition to NGS library preparation, the device and controller are suitable for automating other time-consuming and error-prone laboratory protocols requiring column chromatography steps, such as chromatin immunoprecipitation.

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