If you are like most of us at PacBio, you likely learned how to extract DNA in a high school or college biology class, or maybe even in your kitchen. But as you moved on to more high-stakes experiments, you may have found that extracting DNA for sequencing in your lab isn’t always as straightforward as lyse, precipitate, wash, suspend. In this introduction to DNA extraction, we will share tips, tricks, and protocols to help make your DNA isolation easier.
For optimal results to power biological discovery, sample prep is a critical step in any sequencing project. And with long-read sequencing technologies, including HiFi sequencing, you not only want DNA free from nicks and degradation, but you also want long fragments (tens of kilobases) to achieve those coveted long reads.
Long-read sequencing expert and sample wrangler Olga Pettersson (@OlgaVPettersson) of SciLifeLab at Uppsala University, advises: “Aim for getting molecules as long as you can, as pure as you can, as fresh as you can.”
So, what are the factors that go into obtaining HMW DNA for sequencing? Jennifer Balacco (@JenBalacco) of the Vertebrate Genome Lab, which aims to sequence the genomes of all living vertebrate species and therefore has a wealth of experience with DNA extraction, points to sample type, the prep and storage of samples, and individualized extraction methods as the key components of successful DNA extraction. You can follow along as she shares her experience with many sample types in the video below and then explore our additional resources and considerations.
Watch this PacBio Virtual Global Summit presentation from Jennifer Balacco of the Vertebrate Genome Lab on DNA extraction approaches to achieve error-free genomes.
DNA extraction challenge #1: sample type
Both within the same organisms and between species, there is variability in how readily HMW DNA can be extracted and how stable it is once extracted. DNA from liver, for example, is known to quickly degrade due to the enzymes that make a functioning liver, while DNA extracted from blood is typically more stable. Some plant species have phenolics and polysaccharides that interfere with extraction, and mollusks have high DNAase activity that makes it difficult to store DNA for any amount of time.
If you have a choice on the type of sample you use, a cell-dense tissue with minimal potential contaminants is your best bet. For vertebrates, this means using tissues like blood, brain, kidney, or muscle. For some invertebrates there may be a mucous membrane that inhibits the ability to obtain high-quality DNA, and you might want to consider an additional DNA cleanup step to rid the extraction of contaminants.
When working with small arthropods, you can use an adult individual but may find that targeting pupae or larva are an easier DNA source than a tough exoskeleton-covered adult. When planning a fungus sequencing project, consider culturing the sample in order to acquire a single isolate/individual in the case of macroscopic organisms or an isogenic population in the case of microorganisms. And finally, for plants, it is recommended to obtain the youngest leaf/shoot tissue from an individual plant that has been dark treated (kept out of light) for 24–72 hours.
DNA extraction challenge #2: sample prep and storage
The second consideration for HMW DNA extraction after you’ve decided what sample type to use is how you will treat that sample. Sequencing adheres to the “garbage in, garbage out” rule; therefore it’s prudent to take care when prepping your samples. In most cases, the freshest sample will work best, followed by samples flash frozen with liquid nitrogen and stored at −80°C. This is because as soon as a tissue is taken from its living organism it begins to release factors that degrade both DNA and RNA, making it a race against the clock to get the genetic material out intact.
“Aim for getting molecules as long as you can, as pure as you can, as fresh as you can.”
— Olga Pettersson, SciLifeLab at Uppsala University
Of course, we can’t always control how a sample is prepped or stored, and in those cases it’s generally worth a try to get the best DNA you can from any given sample. There are examples of ethanol stored samples providing sufficient quality DNA as well as museum specimens for amplicon sequencing. However you decide to prep and store your sample prior to DNA extraction, the main aim is to reduce the amount of time between sampling and stably storing your sample to reduce enzymatic degradation of the genetic material within.
DNA extraction challenge #3: choosing the right method
The final piece of the puzzle when it comes to obtaining HMW DNA for a sequencing project is the method used for extraction. There is no shortage of kits, protocols, and tutorials for DNA extraction, and after spending years trying to find the best one-size-fits-all extraction method for various sample types, we are fairly confident one doesn’t exist! However, there are some approaches that consistently produce plentiful HMW DNA that can be binned by sample type.
In general, “old school” methods using chemicals commonly found in molecular biology labs perform fairly well. For example, phenol and chloroform extractions work well for many tissues, though the chemicals used are dangerous. The cetyl trimethylammonium bromide (CTAB) method for extraction of DNA from plants is also a fairly robust way to yield good DNA. And once you understand the chemistry of how DNA is liberated from cells via these methods, you can tailor the protocols to meet the needs of individual species.
If you’re in the market for a tailored protocol, we encourage you to check out Extract DNA for PacBio, where we have collected many protocols from published projects, organized by organism type. However, if you’re looking for an easy, all-in-one DNA extraction kit to get you started on your sequencing journey, there are a few out there that have produced great DNA for HiFi sequencing, and are summarized in our DNA extraction technical note. If you are hoping to outsource this step to a DNA extraction lab, explore our certified service providers, many of which offer DNA extraction as a service.
While there might not be a one-size-fits-all solution for extracting DNA, we hope our experience and those of our customers can help point you in the right direction for a successful HiFi sequencing project.
If you are ready to get started with sequencing or simply need help with choosing the best DNA extraction approach, connect with a PacBio scientist.
Explore other posts in the Sequencing 101 series:
The evolution of DNA sequencing tools
Introduction to PacBio sequencing and the Sequel II system
Why are long reads important for studying viral genomes?
What’s the value of sequencing full-length RNA transcripts?
Looking beyond the single reference genome to a pangenome for every species
Understanding accuracy in DNA sequencing
From DNA to discovery — the steps of SMRT sequencing