Microfluidic Platform for Next-Generation Sequencing Library Preparation with Low-Input Samples

Abstract

Advances in Next Generation Sequencing (NGS) have made available a wealth of information that had previously been inaccessible to researchers and clinicians. NGS has been applied to understand genomic, transcriptomic, and epigenomic changes and gained traction as a significant tool capable of accelerating diagnosis, prognosis, and biomarker discovery. However, these NGS assays have yet to be practical methods for patient stratification or diagnosis because of the gap between the tiny quantities of biomaterials provided by a clinical sample and the large DNA input required by most of these assays. Current library preparation methodologies typically require large input amounts of DNA and a long and complicated manual process. Here, we present a microfluidic droplet-based system for NGS library preparation, capable of reducing the number of pipetting steps significantly, reducing reagent consumption by 10x, automating much of the process, while supporting extremely low DNA input requirement (10 pg per library). This semiautomated technology will allow for low-input preparations of 8 libraries simultaneously while reducing batch-to-batch variation and operator hands-on time.

Target Enrichment Approaches for Next-Generation Sequencing Applications in Oncology

“…This technology provides the advantages of minimizing the DNA and reagent requirement to nanoliters along with automation and consolidation of the PCR reaction onto one nanofluidic chip. Post-PCR, the amplicons from each sample can be retrieved from the reaction compartments and processed further for sequencing [ 21 , 22 ]. Another specialized PCR technology that has been adapted for NGS target enrichment is anchored multiplex PCR, where the amplification is open-ended: only one side of the ROI sequence is targeted using a target-specific primer (anchor), while the other end is targeted with a universal primer.…”

Cilo-seq: highly sensitive cell-in-library-out single-cell transcriptome sequencing with digital microfluidics

“…This has resulted in reproducible sequencing libraries, allowing integration of complex library preparation and purification processes in a single device. 19 Unfortunately, until now, a fully integrated scRNA-seq platform allowing for single-cell isolation and lysis, cDNA generation and amplification, library preparation and purification in the same system is still lacking. Multiple-step pipettings/transfers results in nucleic acid loss and random errors, thus compromising the detection sensitivity and accuracy.…”

Toward nanofluidics‐based mass spectrometry for exploring the unknown complex and heterogeneous subcellular worlds

“…Nanofluidics is the study and application of fluids confined within nanostructures. [20][21][22][23][24]41 With the advancement of nanofab-rication over the past two decades, [25][26][27][28][29][30][31][32][33]34,35 nanostructures with various nanofluidic geometries such as nanopipettes, nanotubes, nanopores, and nanochannels have been fabricated, resulting in diverse abilities and great potential for a variety of applications. In particular, the use of these nanofluidic geometries allows the precise handling of fluid samples with ultrasmall volumes ranging from picoliter to zeptoliter (10 −21 L, zl) order.…”