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Next Generation Sequencing (NGS), or deep sequencing, provides researchers and clinicians with extensive genetic data with a multitude of applications. By fragmenting genetic materials and performing millions to billions of reads simultaneously, next generation sequencing is faster, more reliable, and lower cost than traditional sequencing methods. However, NGS applications can be limited by sample size and quality. In this article, learn how plasma treatment is used in conjunction with next generation sequencing to optimize results.
Effective sample preparation is integral to obtaining high-quality, accurate next generation sequencing results. While the type of genetic material used in next generation sequencing varies widely, often the initial goal is to isolate a homologues sample pool. Acquiring a sample set with high purity is essential for avoiding bias. This task can be incredibly difficult for clinical samples, where target cell availability is low. New sample preparation methods are needed to generate high quality, and pure NGS libraries with low input.
One promising avenue for cell isolation or DNA/RNA purification are microfluidic devices fabricated by plasma treatment. Microfluidics offer precise control over cells and biomolecules. Using physical or chemical controls, researchers can generate pure sample pools with high accuracy. Custom microfluidic devices can be rapidly produces at a low cost using PDMS bonding via plasma treatment.
Featured Next Generation Sequencing Articles
Murphy T, Hsieh Y, Zhu B, Naler L, and Lu C. “Microfluidic platform for next-generation sequencing library preparation with low-input samples”.
Analytical Chemistry 2020 92: 2519-2526 10.1021/acs.analchem.9b04086
Oncology: Circulating Tumor cell (CTC) Isolation
An area of intense focus for next generation sequencing is oncology, where individual genetic sequencing may impact patient health most dramatically. Researchers are developing new methods for personalized diagnosis, prognosis, and drug discovery. A key step in many methods is isolating target cells from patient samples, often circulating tumor cells (CTCs). However, isolating CTC’s with enough genetic material for useful sequencing practices is difficult, particularly in early stage cancer. Microfluidic devices offer promising results for isolating small samples that can be used with next generation sequencing requirements. In several of these cases, on chip assays can be used in conjunction with NGS to provide even more substantial results.
- Centerfuge Chip [Che et al]
- Lateral Magnetophoresis [Cho et al]; [Kang et al]
- Droplet microfluidics [Liu et al]; [Wong et al]
- Microfluidic Ratchet (size and deformability) [Park et al]
In the following articles, microfluidic devices are constructed by PDMS bonding with Harrick Plasma cleaners. These microfluidic devices are designed to isolate CTCs that may later be used for next generation sequencing.
CTC Isolation Research Articles
Che J, Mach AJ, Go DE, Talati I, Ying Y, Rao J, Kulkarni RP, and Di Carlo D. “Microfluidic Purification and Concentration of Malignant Pleural Effusions for Improved Molecular and Cytomorphological Diagnostics”. PLoS One 2013 8: e78194 10.1371/journal.pone.0078194
Cho H, Kim J, Jeon C-W, and Han K-H. “A disposable microfluidic device with a reusable magnetophoretic functional substrate for isolation of circulating tumor cells”. Lab. Chip 2017 17: 4113-4123 10.1039/C7LC00925A
Kang H, Kim J, Cho H and Han K. “Evaluation of Positive and Negative Methods for Isolation of Circulating Tumor Cells by Lateral Magnetophoresis”, Micromachines 2019 10 10.3390/mi10060386
Liu W. “Improving Disease Diagnostics using Microfluidics”. University of Toronto 2020 http://hdl.handle.net/1807/100963
Park ES, Duffy SP, and Ma H. “Microfluidic Separation of Circulating Tumor Cells Based on Size and Deformability”. Methods Mol. Biol. 2017 1634: 21-32 10.1007/978-1-4939-7144-2_2
Wong AH-H, Li H, Jia Y, Mak P-I, Martins RPdS, Liu Y, Vong CM, Wong HC, Wong PK, Wang H, Sun H, and Deng C-X. “Drug screening of cancer cell lines and human primary tumors using droplet microfluidics”. Sci. Rep. 2017 7: 9109 10.1038/s41598-017-08831-z
DNA (or RNA) Purification
Following DNA or RNA extraction from a target cell population, further purification steps may be necessary prior to NGS library generation. In several next generation platforms, a specific DNA or RNA size range is needed for effective sequencing. Again, the control offered by microfluidic devices over biomolecules lends itself to optimal NGS results.
In the following articles, oligomers are sorted by size using microfluidic devices for use in next generation sequencing.
DNA (or RNA) Purification Research Articles
Han C, Catoe D, Munro S, Khnouf R et al “Simultaneous RNA purification and size selection using on-chip isotachophoresis with an ionic spacer”. Lab on a Chip 2019 19: 2741-2749 10.1039/C9LC00311H
Jones PV, Salmon GL, and Ros A. “Continuous Separation of DNA Molecules by Size Using Insulator-Based Dielectrophoresis”. Anal. Chem. 2017 89: 1531-1539 10.1021/acs.analchem.6b03369
Immobilizing DNA or RNA on substrate surfaces is an important step in the development of next generation sequencing platforms, particularly sequencing by synthesis. In NGS flow cells, oligomers are positioned to hybridize with incoming genetic material, for cluster generation and analysis. Plasma treatment chemically modifies substrates through the introduction of polar functional groups. This hydrophilicity surface can either directly bind biomolecules or can facilitate deposition of coatings, such as silanization.
In the following articles, plasma surface modification is used to immobilize or manipulate biomolecules for use in next generation sequencing.
Biomolecule Immobilization Research Articles
Bose S, Wan Z, Carr A, Rizvi AH, Vieira G, Pe’er D, and Sims PA. “Scalable microfluidics for single-cell RNA printing and sequencing”. Genome Biol. 2015 16: 1 10.1186/s13059-015-0684-3
Lim H, Oliver P, Marzillier J, and Vezenov D. “Heterobifunctional modification of DNA for conjugation to solid surfaces”. Anal. Bioanal.Chem. 2010 397: 1861–1872 10.1007/s00216-010-3733-5
Nelson EM, Li H, and Timp G. “Direct, Concurrent Measurements of the Forces and Currents Affecting DNA in a Nanopore with Comparable Topography”. ACS Nano 2014 8: 5484–5493 10.1021/nn405331t