title: Quality control of next-generation sequencing library through an integrative digital microfluidic platform.
abstract: We have developed an automated quality control (QC) platform for next-generation sequencing (NGS) library characterization by integrating a droplet-based digital microfluidic (DMF) system with a capillary-based reagent delivery unit and a quantitative CE module. Using an in-plane capillary-DMF interface, a prepared sample droplet was actuated into position between the ground electrode and the inlet of the separation capillary to complete the circuit for an electrokinetic injection. Using a DNA ladder as an internal standard, the CE module with a compact LIF detector was capable of detecting dsDNA in the range of 5-100 pg/μL, suitable for the amount of DNA required by the Illumina Genome Analyzer sequencing platform. This DMF-CE platform consumes tenfold less sample volume than the current Agilent BioAnalyzer QC technique, preserving precious sample while providing necessary sensitivity and accuracy for optimal sequencing performance. The ability of this microfluidic system to validate NGS library preparation was demonstrated by examining the effects of limited-cycle PCR amplification on the size distribution and the yield of Illumina-compatible libraries, demonstrating that as few as ten cycles of PCR bias the size distribution of the library toward undesirable larger fragments.
authors.affiliation: Biotechnology and Bioengineering Department, Sandia National Laboratories, Livermore, CA 94550, USA.
authors.forename: Numrin
authors.initials: N
authors.lastname: Thaitrong
authors.forename: Hanyoup
authors.initials: H
authors.lastname: Kim
authors.forename: Ronald F
authors.initials: RF
authors.lastname: Renzi
authors.forename: Michael S
authors.initials: MS
authors.lastname: Bartsch
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Meagher
authors.forename: Kamlesh D
authors.initials: KD
authors.lastname: Patel
mesh_terms: D004247:DNA / Q000032:analysis / Q000737:chemistry; D046209:Electrophoresis, Microchip / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D015723:Gene Library; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D057230:Limit of Detection; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods / Q000592:standards
chemical_list: D004247:DNA