New DNA Extraction Method for Long-Read Sequencing in Plant Tissues
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Studying the genome of commercially valuable plants like papayas can lead to varieties with stronger disease resistance, higher drought tolerance and other improvements. A protocol developed at the University of Illinois Urbana-Champaign enables labs to isolate high molecular weight (HMW) DNA from a variety of plant species cost-effectively and rapidly.
Novel genome assemblies for plants
Deciphering an organism's genetic code from scratch can provide insights that ultimately make for a better plant. The problem with de novo genome assemblies for plants, however, is that they contain many repetitive DNA elements. This can make it virtually impossible to obtain a good assembly when sequencing short DNA fragments, but longer fragments are able to resolve these repeats.
Together with Ray Ming, Alvaro Hernandez and members of their research teams at the University of Illinois Urbana-Champaign, I recently published a unique DNA extraction method in the journal Cell. It was designed for the preparation of high-purity, HMW plant DNA suitable for long-read next-generation sequencing (NGS).
The method will be used to generate long- and ultralong-reads, which can then be used for the novel genome assembly of Caricaceae species, a family of tropical flowering plants including papaya and other edible fruits. The genome assembly will provide information that furthers the study of this economically important plant family’s sex determination pathways and its evolutionary history.
High molecular weight DNA from plant tissues
DNA quantity and quality are important factors for success in long-read NGS, but achieving adequate levels of both can be challenging. Because it was necessary to generate long DNA fragments, our research team knew we needed to develop a procedure specific to plant tissues.
Starting with the right sample was important and led to improved HMW DNA purity. We found that using fresh, young leaves without any signs of disease preferable because adult leaves can contain a higher level of contaminants like polysaccharides, polyphenols and others.
In addition to using fresh leaves for sampling, we implemented a purification step before sequencing to address any residual impurities such as proteins, phenols or other organic compounds.
Gentle handling and the use of specialized tools, such as wide-bore pipette tips, throughout the workflow were also key in preventing DNA fragmentation during preparation. Fragmentation would ultimately lead to low yields of suitable length DNA for long-read or ultralong-read DNA sequencing.
The quality and quantity of the extracted DNA were then tested using various quality assessments.
Multiple quality tests with different goals
By using sample quality control procedures, the team was able to ensure that the DNA extractions contained fragments that were highly pure and of sufficient length for long- and ultralong-read sequencing. The team used ultraviolet-visible (UV-Vis) spectrophotometry and fluorometry to measure DNA quantity and identify contaminants. We verified the presence of long fragments in the samples by running the DNA on an agarose gel.
Although we could detect long DNA fragments, ultralong DNA fragments and large quantities of small DNA fragments on the agarose gel, it was difficult to see small quantities of shorter DNA fragments under 50 kilobases.
To improve the discernment of small fragments in the DNA samples, we used a fragment analyzer system. The system presents data in an electropherogram, which makes it easy to see small quantities of shorter fragments, even if those short fragments are spread out. This analysis made it possible for us to determine whether a sample was of acceptable quality and length for long-read sequencing. The results confirmed that our DNA extraction method was performing as intended.
Just the beginning for plant genome assembly
Our extraction method, as validated by distinct quality control techniques, can produce high-purity samples that yield an average read distribution of 25 kilobases. These reads can then be used for novel genome assemblies of different species in the Caricaceae family. Using this method allows in-house extractions to be performed, eliminating the cost of sending samples to service labs.
Although we originally designed this extraction method to obtain suitable DNA from plant tissues for Oxford Nanopore long-read sequencing, we envision that the procedure could also be used with other sequencing platforms, such as PacBio or Illumina sequencing technologies.
We also anticipate that the method will work well for analyzing tissues from other plant species beyond Caricaceae. There are many species that still have not had their genomes assembled, but more analyses like these are emerging for plants every day. By making it simpler and cheaper to generate high-purity HMW DNA from plant tissues, we hope to enable more research in support of a more resilient food system, enhanced prospects for bioenergy and other benefits gained from a deeper understanding of plant biology.
Reference: Zerpa-Catanho D, Zhang X, Song J, Hernandez AG, Ming R. Ultra-long DNA molecule isolation from plant nuclei for ultra-long read genome sequencing. Cell 2021, 183, 875-889.e117. doi:10.1016/j.xpro.2021.100343