A microfluidic DNA library preparation platform for next-generation sequencing - PubMed (original) (raw)

. 2013 Jul 22;8(7):e68988.

doi: 10.1371/journal.pone.0068988. Print 2013.

Mais J Jebrail, Anupama Sinha, Zachary W Bent, Owen D Solberg, Kelly P Williams, Stanley A Langevin, Ronald F Renzi, James L Van De Vreugde, Robert J Meagher, Joseph S Schoeniger, Todd W Lane, Steven S Branda, Michael S Bartsch, Kamlesh D Patel

Affiliations

A microfluidic DNA library preparation platform for next-generation sequencing

Hanyoup Kim et al. PLoS One. 2013.

Abstract

Next-generation sequencing (NGS) is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with >99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM). The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Nextera® protocol for preparing DNA libraries compatible with Illumina sequencers.

Key steps include: gDNA tagmentation, clean-up, limited-cycle PCR, and selection of size-specific DNA library for sequencing and analysis.

Figure 2

Figure 2. Integrated microfluidic system for preparing DNA libraries for sequencing.

a) Top view of an integrated system and side view of the DMF-capillary interface, which features a central DMF hub for integrating multiple reagent and sample preparation modules (depicted in different colors), magnets and thermal blocks coupled to module tubing for sample preparation, and multi-valve syringe pumps for liquid handling. b) Schematic (left) of a DMF device showing the arrangement of indium tin oxide (ITO) actuation electrodes used to route samples and reagents to and from the capillaries, which are responsible for transferring liquids to their respective modules. Sequence of frames (right) from a movie illustratfing the stages in preparing a sequencer-ready DNA library using the microfluidic method (note that off-DMF processing is not shown in the perspective of the frames): (1) Mixing of gDNA (1.8 µL) and Nextera Enzyme (NE, 0.6 µL) droplets; (2,3) post-tagmentation droplet (gDNA+NE reaction products) merged with magnetic bead (MB) droplet (4.5 µL) and actuated to clean-up module; (4,5) post-clean-up droplet (2.2 µL) of purified DNA fragments actuated to PCR Mix droplet (2.8 µL) for limited-cycle PCR; (6,7) post-PCR DNA fragment droplet mixed with different volumes (2.2 and 0.45 µL) of magnetic beads for size-selection; and (8) DNA library droplet (3 µL).

Figure 3

Figure 3. Modular format for implementing magnetic bead-based steps.

Series of images from a movie (side-view) depicting key magnetic bead-based steps in an off-DMF module. Blue- and red-dashed lines show the walls of module and air/bolus interface, respectively. (1, 2) An external magnet immobilizes beads onto the tube surface. (3) Positive pressure-driven flow separates supernatant away from the bead pellet. (4, 5) Removing the magnet and shuttling a bolus of solution over the pellet resuspends immobilized beads. Arrows indicate the direction of bolus movement.

Figure 4

Figure 4. Analysis of human gDNA at different stages of the Nextera protocol.

Bioanalyzer traces of a) gDNA, b) post-tagmentation, c) post-limited-cycle PCR and d) post-size-selection. Peaks at 35 and 10380 bp represent low- and high-molecular weight markers.

Figure 5

Figure 5. Genome coverage plots for three E. coli libraries prepared by microfluidic method.

The plot represents relative depth of sequence coverage across the entire ∼4.6 kb E. coli genome.

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Grants and funding

This work was funded by Sandia National Laboratories’ Grand Challenge LDRD (Laboratory-Directed Research and Development, grant number 142042) program. Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin company, for the U.S. Department of Energy’s National Nuclear Security Administration under Contract DE-AC04-94AL85000. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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