A microfluidic DNA library preparation platform for next-generation sequencing

• Published in: PloS one 2013-07, Vol.8 (7), p.e68988-e68988
• Main Authors: Kim, Hanyoup, Jebrail, Mais J, Sinha, Anupama, Bent, Zachary W, Solberg, Owen D, Williams, Kelly P, Langevin, Stanley A, Renzi, Ronald F, Van De Vreugde, James L, Meagher, Robert J, Schoeniger, Joseph S, Lane, Todd W, Branda, Steven S, Bartsch, Michael S, Patel, Kamlesh D
• Format: Article
• Language: English
• Subjects: Automation; Bacteria; BASIC BIOLOGICAL SCIENCES; Bioengineering; Biology; Biotechnology; Chemistry; Deoxyribonucleic acid; DNA; DNA sequencing; DNA, Bacterial - genetics; Drug resistance; E coli; Engineering; Gene Library; Gene sequencing; Genetic testing; Genome, Bacterial - genetics; Genome, Human - genetics; Genomes; Genomic libraries; Genomics; High-Throughput Nucleotide Sequencing - instrumentation; Humans; Immunoassay; Klebsiella; Laboratories; Life sciences; Medicine; Microfluidic Analytical Techniques - instrumentation; Microfluidics; Multidrug resistance; Nucleotide sequence; Protocol; Science & Technology - Other Topics; Sequence Analysis, DNA - instrumentation; Systems Integration; Technology application
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0068988

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.