Microfluidic Acoustic Method for High Yield Extraction of Cell-Free DNA in Low-Volume Plasma Samples.

Cell-free DNA has many applications in clinical medicine, in particular in cancer diagnosis and cancer treatment monitoring. Microfluidic-based solutions could provide solutions for rapid, cheaper, decentralized detection of cell-free tumoral DNA from a simple blood draw, or liquid biopsies, replacing invasive procedures or expensive scans. In this method, we present a simple microfluidic system for the extraction of cell-free DNA from low volume of plasma samples (≤500 µL). The technique is suitable for either static or continuous flow systems and can be used as a stand-alone module or integrated within a lab-on-chip system. The system relies on a simple yet highly versatile bubble-based micromixer module whose custom components can be fabricated with a combination of low-cost rapid prototyping techniques or ordered via widely available 3D-printing services. This system is capable of performing cell-free DNA extractions from small volumes of blood plasma with up to a tenfold increase in capture efficiency when compared to control methods.

Nuclease Enrichment and qPCR Detection of Rare Nucleotide Variants.

The emergence of circulating DNA analysis in blood during the past decade has responded to the need for noninvasive alternatives to classical tissue biopsies. This has coincided with the development of techniques that allow the detection of low-frequency allele variants in clinical samples that typically carry very low amounts of fragmented DNA, such as plasma or FFPE samples. Enrichment of rare variants by nuclease-assisted mutant allele enrichment with overlapping probes (NaME-PrO) enables a more sensitive detection of mutations in tissue biopsy samples alongside standard qPCR detection assays. Such sensitivity is normally achieved by other more complex PCR methods, such as TaqMan qPCR and digital droplet PCR (ddPCR). Here we describe a workflow of mutation-specific nuclease-based enrichment combined with a SYBR Green real-time quantitative PCR detection method that provides comparable results to ddPCR. Using a PIK3CA mutation as an example, this combined workflow enables detection and accurate prediction of initial variant allele fraction in samples with a low mutant allele frequency (<1%) and could be applied flexibly to detect other mutations of interest.

A method for multiplexed full-length single-molecule sequencing of the human mitochondrial genome.

Methods to reconstruct the mitochondrial DNA (mtDNA) sequence using short-read sequencing come with an inherent bias due to amplification and mapping. They can fail to determine the phase of variants, to capture multiple deletions and to cover the mitochondrial genome evenly. Here we describe a method to target, multiplex and sequence at high coverage full-length human mitochondrial genomes as native single-molecules, utilizing the RNA-guided DNA endonuclease Cas9. Combining Cas9 induced breaks, that define the mtDNA beginning and end of the sequencing reads, as barcodes, we achieve high demultiplexing specificity and delineation of the full-length of the mtDNA, regardless of the structural variant pattern. The long-read sequencing data is analysed with a pipeline where our custom-developed software, baldur, efficiently detects single nucleotide heteroplasmy to below 1%, physically determines phase and can accurately disentangle complex deletions. Our workflow is a tool for studying mtDNA variation and will accelerate mitochondrial research.

Engineered Membranes for Residual Cell Trapping on Microfluidic Blood Plasma Separation Systems: A Comparison between Porous and Nanofibrous Membranes.

Blood-based clinical diagnostics require challenging limit-of-detection for low abundance, circulating molecules in plasma. Micro-scale blood plasma separation (BPS) has achieved remarkable results in terms of plasma yield or purity, but rarely achieving both at the same time. Here, we proposed the first use of electrospun polylactic-acid (PLA) membranes as filters to remove residual cell population from continuous hydrodynamic-BPS devices. The membranes hydrophilicity was improved by adopting a wet chemistry approach via surface aminolysis as demonstrated through Fourier Transform Infrared Spectroscopy and Water Contact Angle analysis. The usability of PLA-membranes was assessed through degradation measurements at extreme pH values. Plasma purity and hemolysis were evaluated on plasma samples with residual red blood cell content (1, 3, 5% hematocrit) corresponding to output from existing hydrodynamic BPS systems. Commercially available membranes for BPS were used as benchmark. Results highlighted that the electrospun membranes are suitable for downstream residual cell removal from blood, permitting the collection of up to 2 mL of pure and low-hemolyzed plasma. Fluorometric DNA quantification revealed that electrospun membranes did not significantly affect the concentration of circulating DNA. PLA-based electrospun membranes can be combined with hydrodynamic BPS in order to achieve high volume plasma separation at over 99% plasma purity.

Correction: Versatile hybrid acoustic micromixer with demonstration of circulating cell-free DNA extraction from sub-ml plasma samples.

Correction for 'Versatile hybrid acoustic micromixer with demonstration of circulating cell-free DNA extraction from sub-ml plasma samples' by Alvaro J. Conde et al., Lab Chip, 2020, 20, 741-748, DOI: 10.1039/C9LC01130G.

PIK3CA mutation enrichment and quantitation from blood and tissue.

PIK3CA is one of the two most frequently mutated genes in breast cancers, occurring in 30-40% of cases. Four frequent 'hotspot' PIK3CA mutations (E542K, E545K, H1047R and H1047L) account for 80-90% of all PIK3CA mutations in human malignancies and represent predictive biomarkers. Here we describe a PIK3CA mutation specific nuclease-based enrichment assay, which combined with a low-cost real-time qPCR detection method, enhances assay detection sensitivity from 5% for E542K and 10% for E545K to 0.6%, and from 5% for H1047R to 0.3%. Moreover, we present a novel flexible prediction method to calculate initial mutant allele frequency in tissue biopsy and blood samples with low mutant fraction. These advancements demonstrated a quick, accurate and simple detection and quantitation of PIK3CA mutations in two breast cancer cohorts (first cohort n = 22, second cohort n = 25). Hence this simple, versatile and informative workflow could be applicable for routine diagnostic testing where quantitative results are essential, e.g. disease monitoring subject to validation in a substantial future study.

Versatile hybrid acoustic micromixer with demonstration of circulating cell-free DNA extraction from sub-ml plasma samples.

Acoustic micromixers have attracted considerable attention in the last years since they can deliver high mixing efficiencies without the need for movable components. However, their adoption in the academic and industrial microfluidics community has been limited, possibly due to the reduced flexibility and accessibility of previous designs since most of them are application-specific and fabricated with techniques that are expensive, not widely available and difficult to integrate with other manufacturing technologies. In this work, we describe a simple, yet highly versatile, bubble-based micromixer module fabricated with a combination of low-cost rapid prototyping techniques. The hybrid approach enables the integration of the module into practically any substrate and the individual control of multiple micromixers embedded within the same monolithic chip. The module can operate under static and continuous flow conditions showing enhanced mixing capabilities compared to similar devices. We show that the system is capable of performing cell-free DNA extractions from small volumes of blood plasma (≤500 µl) with up to a ten-fold increase in capture efficiency when compared to control methods.

A simple and robust real-time qPCR method for the detection of PIK3CA mutations.

PIK3CA mutations are seemingly the most common driver mutations in breast cancer with H1047R and E545K being the most common of these, accounting together for around 60% of all PIK3CA mutations and have promising therapeutic implications. Given the low sensitivity and the high cost of current genotyping methods we sought to develop fast, simple and inexpensive assays for PIK3CA H1047R and E545K mutation screening in clinical material. The methods we describe are based on a real-time PCR including a mutation specific primer combined with a non-productive oligonucleotide which inhibits wild-type amplification and a parallel internal control reaction. We demonstrate consistent detection of PIK3CA H1047R mutant DNA in genomic DNA extracted from frozen breast cancer biopsies, FFPE material or cancer cell lines with a detection sensitivity of approximately 5% mutant allele fraction and validate these results using both Sanger sequencing and deep next generation sequencing methods. The detection sensitivity for PIK3CA E545K mutation was approximately 10%. We propose these methods as simple, fast and inexpensive diagnostic tools to determine PIK3CA mutation status.