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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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Related in: MedlinePlus

Coverage depth from a sequencing run using an Illumina U-2 OS osteosarcoma cell line library prepared on the AMCC chip.The library was run on the MiSeq using the 2x150 bp paired-end sequencing protocol. (a) Distribution of sequencing reads across the reference human genome, which has been divided into 1 million bins to assess coverage uniformity. (b) Average coverage across different chromosomes. (c) Average sequencing depth across different chromosomes. The Y chromosome is absent from the U-2 OS osteosarcoma cell line.
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pone-0064084-g004: Coverage depth from a sequencing run using an Illumina U-2 OS osteosarcoma cell line library prepared on the AMCC chip.The library was run on the MiSeq using the 2x150 bp paired-end sequencing protocol. (a) Distribution of sequencing reads across the reference human genome, which has been divided into 1 million bins to assess coverage uniformity. (b) Average coverage across different chromosomes. (c) Average sequencing depth across different chromosomes. The Y chromosome is absent from the U-2 OS osteosarcoma cell line.

Mentions: Using the 2 by 150 bp sequencing kit on the MiSeq, we generated approximately 10.5 million reads each for reads 1 and 2 with the elute from reactor 7. Sequencing data of approximately 3 gigabases were mapped to the reference human genome (hg19) with an alignment rate of 99.37%. To evaluate if the sequencing library provided uniform coverage of the genome, we divided the human reference genome into 1 million bins (each 3096 bp long) across all chromosomes (Figure 4A). 92.7% of the bins overlapped at least one of the sequencing reads. There was little variation in sequencing coverage and depth across different chromosomes (Figure 4B and 4C). with an average sequencing coverage of 24.7% (standard deviation 3.9% and an average sequencing depth of 0.61× (standard deviation 0.12×). These data, based on a sampling from a single sequencing run on the MiSeq, suggest that the library provided uniform coverage of the human genome and would be suitable for full-scale sequencing on a higher capacity Illumina platform such as the GAII or HiSeq.


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)

Coverage depth from a sequencing run using an Illumina U-2 OS osteosarcoma cell line library prepared on the AMCC chip.The library was run on the MiSeq using the 2x150 bp paired-end sequencing protocol. (a) Distribution of sequencing reads across the reference human genome, which has been divided into 1 million bins to assess coverage uniformity. (b) Average coverage across different chromosomes. (c) Average sequencing depth across different chromosomes. The Y chromosome is absent from the U-2 OS osteosarcoma cell line.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3722208&req=5

pone-0064084-g004: Coverage depth from a sequencing run using an Illumina U-2 OS osteosarcoma cell line library prepared on the AMCC chip.The library was run on the MiSeq using the 2x150 bp paired-end sequencing protocol. (a) Distribution of sequencing reads across the reference human genome, which has been divided into 1 million bins to assess coverage uniformity. (b) Average coverage across different chromosomes. (c) Average sequencing depth across different chromosomes. The Y chromosome is absent from the U-2 OS osteosarcoma cell line.
Mentions: Using the 2 by 150 bp sequencing kit on the MiSeq, we generated approximately 10.5 million reads each for reads 1 and 2 with the elute from reactor 7. Sequencing data of approximately 3 gigabases were mapped to the reference human genome (hg19) with an alignment rate of 99.37%. To evaluate if the sequencing library provided uniform coverage of the genome, we divided the human reference genome into 1 million bins (each 3096 bp long) across all chromosomes (Figure 4A). 92.7% of the bins overlapped at least one of the sequencing reads. There was little variation in sequencing coverage and depth across different chromosomes (Figure 4B and 4C). with an average sequencing coverage of 24.7% (standard deviation 3.9% and an average sequencing depth of 0.61× (standard deviation 0.12×). These data, based on a sampling from a single sequencing run on the MiSeq, suggest that the library provided uniform coverage of the human genome and would be suitable for full-scale sequencing on a higher capacity Illumina platform such as the GAII or HiSeq.

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.

Show MeSH
Related in: MedlinePlus