Varuni Ankolekar, Quartesian
Genome sequencing has helped a cell to disclose much regarding the biological world. It’s a known fact that the short pieces of DNA, ‘Genes’ are the basic unit of inheritance. They are the reason for the development of different traits in organisms. The differences and similarities between organisms help to understand how the organisms emerged and developed while discovering entirely new species as well. The nucleotide sequences in DNA or RNA code the synthesis of either RNA or protein.
All genetic material in an organism is together known as Genome and the study of the genome is known as Genomics. It includes coding and noncoding DNA, mitochondrial DNA, and chloroplast DNA.
What are genome sequencing and its importance?
Genome sequencing is discovering the sequence of nucleotides or bases in the genome.
Genome sequencing is significant as it reveals where these genes are and how they work collectively to develop, regulate, and maintain the functions in organisms.
Methods of Genome sequencing:
DNA sequencing is done by dividing it into short pieces and putting them back together. Conventional methods have low throughput, large sample consumption with false positive (or negative) errors when complex biological samples were used.
Droplet-based microfluidic systems make use of compartmentalized aqueous droplets by manipulating fluids into immiscible phases. It overcomes the limitation of traditional methods as they reduce large fluid consumption into droplets. The volume of each droplet ranges to the nano-to-femtoliter scale.
There are 3 types: continuous-flow emulsion-based platforms, electrowetting-based platforms, and static microdroplet arrays.
Addressable dynamic droplet array (aDDA):
The benefits of static droplet arrays and continuous-flow droplet platforms
In Static droplet arrays, the identification of the target single cell is accurate while the droplets containing the sample can be retrieved. In continuous-flow droplet platforms unlike static droplet assays processing of each step is continuous and quick. The benefits of both were combined in an Addressable dynamic droplet array (aDDA).
Genome sequencing using this approach was performed in Single Yeast Cells. The sample preparation steps include:
- Isolation of Single Cell
- Lysis
- Multiple displacement amplification (MDA): A technique used to amplify DNA.
- Retrieval of sample
The single-cell isolation, cell lysis, amplification, and retrieval of the product were executed in a sub-nanoliter (≈300 pL) droplet.
Other steps and materials used include:
- Degassed PDMS (Polydimethylsiloxane) pump: Used as a power source to generate monodispersed droplets under reduced risk of external contamination.
- Hydraulic resistance influenced droplet size which was regulated by varied connection tubing between the components.
- Creation of hydrophilic pattern surrounding the PDMS microwells: Microwells were altered by graft polymerization to form hydrophilic patterns.
- Individual droplets from the aDDA are retrieved by peeling the upper layer of the aDDA. Pure mineral oil is utilized to mask the static droplet array to avoid evaporation of droplets and then is carefully picked using customized glass needles.
- PDMS chip is permeable to water allowing water molecules to transfer into the system. Hence, was treated with a steam bath to make it water-saturated.
Multiple displacement amplification (MDA) has a limitation of biased amplification which is reduced by aDDA. The percentage recovery of the single-cell yeast genome is 91%, while 26% is observed in traditional, 20 µL volume MDA reactions.
Thus, aDDA could be one of the robust methods of high-genome-coverage sequencing of single microbial cells.
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References:
1. Li, C., Gong, Y., Wang, X., Xu, J., & Ma, B. (2021). Integrated addressable dynamic droplet array (Adda) as sub‐nanoliter reactors for high‐coverage genome sequencing of single yeast cells. Small, 2100325. https://doi.org/10.1002/smll.202100325
2. What is a gene?: MedlinePlus Genetics. (n.d.). Retrieved August 8, 2021, from https://medlineplus.gov/genetics/understanding/basics/gene/
3. What’s a Genome? (n.d.). Retrieved August 8, 2021, from http://www.genomenewsnetwork.org/resources/whats_a_genome/Chp2_1.shtml
4. Danielson, C., Pappas, G., Phelps, L., Melvin, A. T., & Park, K. (2019). Static microdroplet array generated by spraying and analyzed with automated microscopy and image processing. Analytical Biochemistry, 587, 113452. https://doi.org/10.1016/j.ab.2019.113452
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Author’s Info:
Varuni Ankolekar, Quartesian
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