The process can be performed primarily using mRNA extracts of cells to determine the level of gene expression. For effective RNA-sequencing at the level of the single cell, life scientists must compartmentalize cells in a volume of comparable size to the cell itself. This process can be accomplished in the lab using a microfluidic device. Nevertheless, an existing demand remains to establish a simpler practical method to extend sequencing to a greater number of cells in parallel, while providing single-cell resolution. Preceding methods for cell profiling were only applicable to hundreds or a few thousand cells. As a result, scientists have developed droplet microfluidics as a fundamental method to meet the demand for fast and scalable approaches.

In droplet-based single-cell sequencing, individual cells are compartmentalized in nanolitre-scale aqueous environments that act as tiny reaction chambers for PCR and for reverse transcription. The method can analyze thousands of cells in parallel for cellular mRNA transcripts while recording the transcript’s cell of origin using a genomic barcodes strategy. The barcodes provide a molecular memory, acting as an identifier- read at sequencing to decode specific information of each individual cell. Two recent papers by A. Klein et al. and E. Macosko et al. introduce the technique of droplet microfluidics for single-cell RNA-sequencing to establish the basic concepts (Video 1). Several commercial kits have rapidly followed the protocols and similar publications, to provide easy access to the technique in the lab.

Video 1: Trimmed video abstract of microfluidics-based nanoliter droplet formation for highly parallel genome-wide expression profiling of individual cells. Credit: Cell, DOI: https://doi.org/10.1016/j.cell.2015.05.002

The field is advancing rapidly, with many studies aiming to gather data and explore cellular information accessed with the technique. Microfluidics platforms have advanced to integrate within lab-on-a-chip devices to streamline the process at the nanoscale and dissect transient transcriptional states at the level of the single-nucleus. To achieve higher accuracy in the method, scientists have created droplet libraries that can correct composition bias and sequencing errors affecting cellular and molecular barcodes. The sequencing methods can be extended to probe information beyond mRNA as well. For instance, Guo et al. introduced ultra-high throughput, PCR-free, single-cell miRNA assays in a continuous flow microfluidic process. Droplet-based microfluidics has in this way originated major advances in the field at the interface between technological advancements and biomedical applications of single-cell sequencing.

Fundamental steps of the droplet-based single-cell sequencing method can be summarized as follows:

1. The process starts with the preparation of a single-cell suspension from a tissue of interest in the lab.
2. Scientists then prepare the barcoded primers as surface attachments on simple microparticles or embedded within hydrogel microparticles.
3. A microfluidic device is used to encapsulate individual cells in parallel with a distinctly barcoded microparticle within a tiny droplet.
4. After the cells are isolated in droplets, the cells are lysed to release the mRNAs, which hybridize to the primers.
5. The scientists break the droplets to generate single-cell transcriptomes attached to the microparticles (STAMPs).
6. They amplify the STAMPs with PCR, sequence and analyze the transcript’s cell of origin using the STAMP barcodes.

Two types of beads (microparticles); known as simple and hydrogel can be used to synthesize primers. Each microparticle contains a sequence with a PCR handle, cell barcode, a unique molecular identifier (UMI) and a 30-base pair oligodT sequence at the end of all primer sequences. After preparing the single-cell suspension and the microparticles, the individual cells can be encapsulated together with microparticles in droplets, using a custom-designed intricate microfluidic device. Recent work by life scientists in the McCarroll lab detail the individual steps to synthesize cell-barcodes and UMIs in drop-sequencing, where UMIs act as unique identifiers to prevent double-count sequence reads.

As ongoing high-throughput genome sequencing techniques become practically viable, scientists are beginning to use software tools to visualize the expression of data sets. The functions include routine assembly, annotation and display of genomic information as physical maps. For instance, Lohse et al. describe a suite of tools to generate physical maps of plastid and mitochondrial genomes, known as “Organellar Genome Draw”. The freely available web tool with recent upgrades can mainly analyze large-scale, plant-cell derived nucleic acid and protein sequences. In this way, high-throughput, droplet-based single-cell sequencing is rapidly advancing with technical upgrades and accompanying software to broaden scientific understanding of genomics research at the level of the single cell and single nucleus.

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Thamarasee Jeewandara

Thamarasee Jeewandara is an Early Career Scientist and Science Writer, with a Ph.D. in Medicine and Bioengineering from the Sydney Medical School, University of Sydney, Australia. She is currently living in New York, focusing on her research and writing interests in materials science, tissue engineering, and bone research. Find her on Twitter @Jeew333T and her science writing portfolio on Nature Research Bioengineering.

The post Droplet-Based Single-Cell Sequencing appeared first on The MicroFluidic Circle.

As a result, scientists are interested in developing improved methods for single-cell forensic amplification. One such technique is “Monodisperse Microdroplets” an innovative technique where miniature droplet reactors can be produced using state-of-the-art microfluidics technology to allow for high-sensitivity analysis. Such systems can provide reaction volumes in droplets ranging from the femtoliter to the microliter. In the setup, single cells are housed in discrete aqueous droplets surrounded by an immiscible carrier oil to limit cross-contamination. The microfluidic system can control the droplet size, uniformity and the internal content (such as reagent composition and concentration) to engineer a precise microenvironment for the desired experiment. The microfluidics droplet technology can be used to conduct large-scale independent reactions in parallel, to screen millions of single cells for a specific trait or criminal character. The experiments also allow cell heterogeneity probing to detect rare cells, for applications where a perpetrator has a rarer cell type. The described technology can use single-cell PCR (polymerase chain reaction) to analyze DNA from single cells in forensic cases where only minute or dilute quantities of mixed evidence material are available.

Another drawback to the STR gold standard method is its inability to quickly respond with DNA analyses in situations where delays could impede decisions. For instance, in booking stations and at the border crossing, faster-to-results DNA analysis can benefit forensic investigations. A new method known as Rapid DNA technology aims to address this challenge by integrating samples in a combined DNA index system (CODIS). The setup can generate compatible DNA profiles in less than 90 minutes. The “RapidHIT ID 200” systems were developmentally and experimentally validated for applications in the UK National DNA databases where more than 1000 STR profiles were uploaded. The next-generation microfluidics instrument was specifically developed to address needs in such environments necessitating faster-to-results DNA analyses. In the experimental setup, bulk reagents and a capillary electrophoresis module can be housed inside a plastic compartment, resembling a printer toner-like primary cartridge (GlobalFiler^®)for minimum stability of 6 months at 25⁰C. When an STR profile is generated, the “RapidHIT ID” system can transfer the data to a central computer to process and manually review profiles via the “RapidLINK” software (IntegenX).

The system contains a built-in fingerprint reader and camera to authenticate, access and automate tracking. Experiments are designed in accordance with quality assurance standards of the Federal Bureau of Investigation (FBI) and guidelines of the Scientific Working Group for DNA analysis methods. It is possible to use the RapidHIT technique to yield complete and consistent DNA profiles. For example, highly reliable size precision can be acquired for male and female donor samples, even with minute sample quantities of one touch to the cheek, to identify individuals in order facilities, booking stations and for general laboratory use. In this way,next-generation microfluidics platforms in forensic science aim to facilitate automation and high-throughput processing. The techniques will allow minimal sample contamination alongside rapid identification of dilute, rare or minute samples in self-contained microfluidic platforms.

Students experiencing hands-on work in a forensics lab. Credit: Royal Institution

The experimental platforms detailed here were designed to validate quality assurance standards, offering results in a familiar, conventional STR format. The technologies can be transferred from the lab to the industry. Rapidly advancing microfluidic techniques in molecular biology are thus facilitating fast forensics to crossover from fictional labs to real-time practice. The ready accessibility of forensic science and genetics across the years is highlighted by ongoing outreach efforts that can facilitate a hands-on experience in cutting-edge molecular biology, in a forensics lab for young scientists via workshops. Improved sensitivity and selectivity with microfluidics and capillary electrophoresis systems can refine the scientific method for higher precision. Future work will extend to analyze real-world dilute, contaminated and mixed samples (low-abundance and rare materials) within such platforms for forensic investigations.

Enjoyed this article? Don’t forget to share.

Thamarasee Jeewandara

Thamarasee Jeewandara is an Early Career Scientist and Science Writer, with a Ph.D. in Medicine and Bioengineering from the Sydney Medical School, University of Sydney, Australia. She is currently living in New York, focusing on her research and writing interests in materials science, tissue engineering, and bone research. Find her on Twitter @Jeew333T and her science writing portfolio on Nature Research Bioengineering.

The post Forensic DNA Analysis with Microfluidics appeared first on The MicroFluidic Circle.