Interfacing centrifugal microfluidics with linear-oriented 8-tube strips and multichannel pipettes for increased throughput of digital assays.

We present a centrifugal microfluidic cartridge for the eight-fold parallel generation of monodisperse water-in-oil droplets using standard laboratory equipment. The key element is interfacing centrifugal microfluidics with its design based on polar coordinates to the linear structures of standard high-throughput laboratory automation. Centrifugal step emulsification is used to simultaneously generate droplets from eight samples directly into standard 200 µl PCR 8-tube strips. To ensure minimal manual liquid handling, the design of the inlets allows the user to load the samples and the oil via a standard multichannel pipette. Simulation-based design of the cartridge ensures that the performance is consistent in each droplet generation unit despite the varying radial positions that originate from the interface to the linear oriented PCR 8-tube strip and from the integration of linear oriented inlet holes for the multichannel pipettes. Within 10 minutes, sample volumes of 50 µl per droplet generation unit are emulsified at a fixed rotation speed of 960 rpm into 1.47 × 105 monodisperse droplets with a mean diameter of 86 µm. The overall coefficient of variation (CV) of the droplet diameter was below 4%. Feasibility is demonstrated by an exemplary digital droplet polymerase chain reaction (ddPCR) assay which showed high linearity (R2 ≥ 0.999) across all of the eight tubes of the strip.

Automated library preparation for whole genome sequencing by centrifugal microfluidics.

Next generation sequencing is evolving from a research tool into a method applied in diagnostic routine. The complete sequencing workflow includes sample pre-processing, library preparation, sequencing and bioinformatics. High quality in each of these steps is necessary to obtain excellent sequencing results. The tedious and error-prone library preparation poses a significant challenge for smaller laboratories, where high throughput pipetting robots are not cost-effective. Here we present an automated library preparation for whole genome sequencing using centrifugal microfluidics. Two samples can be run per cartridge. Precise metering of reagents allows the required liquid volumes to be reduced by 40% and the amount of sample used by 60%. The functionality of the cartridge is demonstrated with bacteria and DNA extracted from a human FFPE sample. For the bacterial sample, mean sequencing depths from 140 to 183 reads and a coverage of 99.8% of the reference genome were detected. For the human DNA, mean sequencing depths of 4.4-5.7 reads and a coverage of 78.2% of the effective reference genome were observed.

Point-of-Care System for HTLV-1 Proviral Load Quantification by Digital Mediator Displacement LAMP.

This paper presents a universal point-of-care system for fully automated quantification of human T-cell lymphotropic virus type 1 (HTLV-1) proviral load, including genomic RNA, based on digital reverse RNA transcription and c-DNA amplification by MD LAMP (mediator displacement loop-mediated isothermal amplification). A disposable microfluidic LabDisk with pre-stored reagents performs automated nucleic acid extraction, reaction setup, emulsification, reverse transcription, digital DNA amplification, and quantitative fluorogenic endpoint detection with universal reporter molecules. Automated nucleic acid extraction from a suspension of HTLV-1-infected CD4+ T-lymphocytes (MT-2 cells) yielded 8 ± 7 viral nucleic acid copies per MT-2 cell, very similar to the manual reference extraction (7 ± 2 nucleic acid copies). Fully automated sample processing from whole blood spiked with MT-2 cells showed a comparable result of 7 ± 3 copies per MT-2 cell after a run time of two hours and 10 min.

Automation of Amplicon-Based Library Preparation for Next-Generation Sequencing by Centrifugal Microfluidics.

Next-generation sequencing (NGS) has become a mainstream method in bioanalysis. Improvements in sequencing and bioinformatics turned the complex and cumbersome library preparation to the bottleneck in terms of reproducibility and costs in the complete NGS workflow. Here, we introduce an automated library preparation approach based on a generic centrifugal microfluidic cartridge. Multiplex polymerase chain reaction amplification and subsequent cleanup were performed with all reagents prestored on the disk, including cell-line-based DNA as quality control. Exchange of prestored reagents allows applying the cartridge to different target genes. Sequencing of automatically prepared libraries from T-cell receptor and immunoglobulin gene rearrangements in context of lymphoproliferative disorders demonstrated excellent cleanup performance between 91.9 and 99.9% of target DNA reads and successful amplification of all target regions by up to 15 forward primers combined with 4 reverse primers. The fully automated library preparation by centrifugal microfluidics thus offers attractive automation options in diagnostic settings.