Improving DNA Extraction for NGS

One of the earliest steps in preparing a sample for next-generation sequencing (NGS) is DNA extraction—the process of purifying genetic material from an initial tissue sample as a starting point for NGS library preparation. Generally speaking, DNA extraction involves isolating cells from tissue, and then breaking or solubilizing the cells’ plasma and nuclear membranes to release the genetic material. A variety of methods are available, including phenol-based extraction, chloroform-based extraction, spin columns, magnetic beads, and single-cell isolation. While all approaches have different advantages, technological advances in DNA extraction will improve NGS regardless of your chosen method. This article highlights advances in DNA extraction that help to streamline NGS workflows, and offers expert advice on accomplishing it.

Sample quality

Starting with the best possible sample quality always benefits DNA extraction. “While this may sound mundane, I have found that many customers do not appreciate, at least initially, the impact that the starting quality of tissue can have on all downstream aspects,” says Michelle Kim, Senior Staff Scientist at Circulomics (a PacBio company). Even high molecular weight DNA can be extracted from most tissues if the sample is fresh, or if the tissue was well-cared for with immediate freezing or preservation. One exception is mammalian liver tissue, says Kim, “in which DNA is exceptionally vulnerable to tissue-specific nucleases and is quickly degraded once removed from its living environment.”

Kim prefers freezing over chemical preservation, but acknowledges that logistical limitations such as specimen collection during field work make ethanol preservation an easier choice. “In our hands, mammalian tissues preserved in ethanol are fine following ethanol removal treatment,” she says. “But we and many customers have experienced difficulty working with insect samples stored in ethanol, so sample storage and preservation methods ought be carefully considered.”

When choosing a method for DNA extraction, it is important to consider the downstream applications, as well as the advantages and potential drawbacks of the time, labor, and reagents involved when using each method. “In my opinion, a good method for long-read sequencing is one that results in DNA where the bulk of the molecules easily size larger than 100 kb,” says Kim. “This will provide wide latitude for size selection, if necessary, and shearing.” She notes, however, that some methods yielding DNA molecules over 100 kb are labor-intensive, or use toxic reagents, so the importance of all factors must be weighed when choosing a method.

Extraction for low input, FFPE samples

Low input samples, such as a small biopsy, or a rare cell type isolated from formalin-fixed, paraffin-embedded (FFPE) tissue can be challenging to work with—especially when it comes to obtaining a sufficient quantity of nucleic acid to perform NGS. Modifications to DNA extraction protocols, such as using smaller volumes, can help, as can newer methods such as laser capture microdissection, which physically isolates cells of interest. Enni Markkanen and her research group at the Institute of Veterinary Pharmacology and Toxicology, at the University of Zurich, used laser capture microdissection to isolate a specific sub-population of stroma cells—with an aim to compare normal versus cancerous tissues—followed by RNAseq. (While they were extracting RNA instead of DNA, the principles are similar with respect to the challenges of procuring high-quality nucleic acids from a limited cell population.)

Markkanen’s group adapted an ultrasonication protocol to isolate RNA from de-paraffined FFPE tissue samples, attempting several approaches before finding their optimal method. “The one that performs absolutely best in our hands for laser-capture microdissected FFPE tissue is the FFPE tissue extraction using focused ultrasonication,” she says. They found that focused ultrasonication was more effective compared to the conventional LCM-FFPE tissue extraction protocol using proteases only (https://pubmed.ncbi.nlm.nih.gov/28835206/). “[In my lab], the focused ultrasonication really has made a big impact on the ability to extract tiny quantities of RNA from microdissected FFPE tissue,” says Markkanen.

In contrast to bulk extraction protocols adapted for very low input samples, single-cell sequencing relies on a different method for DNA extraction. For single-cell NGS applications, single cells or nuclei are partitioned into individual droplets containing sequencing reagents. But whether for bulk or single-cell sequencing, careful DNA extraction is crucial for successful NGS results. “For single-cell analysis, sample preparation is key to generating high-quality data,” says Zuleyma Peralta, Associate Product Manager for Sample Prep at 10x Genomics. “If the integrity of the cells is lost before starting a single-cell experiment, the data unfortunately will not represent single cells, but will be a bulk-level experiment.”

With this in mind, the best method of sample preparation for single-cell sequencing may in part depend upon the condition and type of your sample. For instance, solid tissues have to be dissociated first, either enzymatically or mechanically, to generate a single-cell suspension. However, frozen samples deserve extra consideration regarding the analysis of cells versus nuclei. “If starting with frozen tissue, we recommend isolating nuclei rather than cells, because the freeze-thaw process will leave many cells without intact membranes, therefore leaking their contents and precluding single-cell analysis,” says Peralta. For cells in culture, adherent cells usually require enzymatic dissociation, followed by a wash to remove free-floating nucleic acids, while cells grown in suspension may only require the latter. Peralta notes several valuable resources for further information about sample-preparation protocols using different sample types, such as 10x Genomics' Demonstrated Protocols, and the Worthington Tissue Dissociation Guide. No matter what type of sample tissue you choose, improvements in nucleic acid extraction are available to help you streamline your NGS workflow.