Nucleic acid isolation for next-generation sequencing
By: Roche Life Science
Posted: February 09, 2016 | Lab Life - Next-Gen Sequencing
Reliable next-generation sequencing (NGS) results and accurate data analysis require the use of high-quality starting materials. Nucleic acid isolation and purification is the first step in your NGS workflow and should not be underestimated. After all, it forms the building blocks for all future studies from your NGS library. Essentially, it's as simple as "you get out what you put in."
In this article, we will discuss the critical factors involved in the extraction of highly pure RNA or DNA from your experimental samples for NGS library preparation.
Extraction and purification techniques
When you're performing next-generation DNA or RNA sequencing, including transcriptome or whole genome sequencing, the isolation of high-quality nucleic acids is essential for efficient and reproducible studies. There are two main workflow categories for nucleic acid purification when performing NGS: manual and automated. Depending on your volume and throughput needs and the variables being tested, your nucleic acid purification needs may vastly differ. The most popular methods for nucleic acid extraction for NGS library preparation are summarized below.
1. Organic extraction
If you are working with small amounts of tissue in lower throughput experiments, then organic extraction with homogenization in a phenol-containing solution may be a reasonable approach for high yield and cost-efficiency. This method is simple to perform and can be readily scaled for larger tissue samples. Indeed, this is the gold standard technique for nucleic acid purification and protocols have been well-established, making this an excellent procedure for novice researchers. Most importantly, it produces high-quality samples for NGS applications. However, there are inherent drawbacks: It is more laborious, less amenable to higher-throughput processing, can be difficult to automate and requires care in managing the chlorinated organic waste.
2. Spin column extraction
Alternatively, you might opt for a spin-column-based extraction for increased efficiency and kit format availability. This method utilizes a solid phase extraction to bind and isolate nucleic acids within filter-based spin columns. It is therefore amenable to RNA or DNA isolation needs. For instance, the Roche High Pure DNA and RNA isolation kits bind nucleic acids to the surface of a glass fiber fleece in the presence of chaotropic salts, while contaminants are removed by centrifugation. This method is ideal for low- to medium-throughput assays and the isolated nucleic acids are of maximum quality for subsequent NGS applications.
The advantages of this system include a straightforward procedure and availability in kit format, which again is great for junior researchers. The ease of the spin filter makes this method highly amenable to large scale and high-throughput processing, including automated methods. This spin columns are compatible with both centrifugation and vacuum-based systems. The downside of this strategy is that the membranes can become clogged when there is too much starting material (such as larger tissue samples) or incomplete homogenization, which can result in lower yields or potential contamination. Moreover, automated centrifugation or vacuum systems can be costly and may involve a complex setup.
3. Magnetic bead extraction
Another kit-based strategy for nucleic acid isolation over a range of throughput needs is magnetic particle extraction. This strategy uses beads with a paramagnetic core coated with a matrix of silica, most commonly for binding nucleic acids. The magnetic beads can be rapidly collected in proximity to an external magnetic field and the magnetic collection and re-suspension steps are quick and easy to perform. Thus, this is the ideal nucleic acid isolation method for automated protocols. Furthermore, the absence of a filter eliminates concern for clogging and there is no organic solvent hazardous waste. The downside is that viscous samples can impede migration of magnetic beads, and users must be cautious not to contaminate samples with residual magnetic beads.
There are a number of commercially available platforms for magnetic bead methods. The Roche MagNA Pure Compact System is an automated benchtop instrument ideal for low- to medium-sample throughput, processing one to eight samples per run. For higher throughput workflows, the MagNA Pure LC 2.0 tabletop instrument uses an automated robotic system to process up to 32 samples per run in as little as one hour. For the utmost in automated workflow efficiency, the Roche MagNA Pure 96 System can process up to 96 samples with a wide range of starting material in less than one hour. The use of barcoded trays with pre-filled and ready-to-use reagents ensures high reproducibility and accuracy, while the resulting high-quality nucleic acids are ideal for a variety of NGS applications.
Types of starting materials
In regards to your starting material, there are two very important factors to consider for your NGS workflow. Namely, you must take into account the type of nucleic acid to be isolated and the material from which you are extracting it. Depending on these variables, there are multiple kit-based systems available for purification.
There are Roche High Pure spin column and MagNA Pure magnetic bead isolation kits available for a broad range of starting materials, such as: mammalian cells and tissues. including human blood plasma or serum; ethanol preserved tissues; formalin-fixed, paraffin-embedded (FFPE) tissues; bacteria; yeast; fungal cells and even viruses.
Whether you choose organic, spin column or magnetic extraction, it is critical to perform appropriate quality control measures to assess the quantity and purity of your extraction and to evaluate for any degradation or contamination prior to NGS.
One approach is to utilize a NanoDrop spectrophotometer and a 0.7 percent agarose gel. Ideally, you should obtain a single 260 nm absorbance peak, a 260/280 absorbance ratio of 1.8-2.0, and minimal shearing or contamination. Alternatively, the Qubit Fluorometer quantifies DNA while the ThermoFisher Ion Sphere™ Quality Control Kit can assess particle quality. Moreover, when purifying RNA, quality can be assessed by determining an RNA integrity or RIN score ranging from zero to 10, using the Agilent 2100 Bioanalyzer to obtain quality information about your RNA sample. But no matter the method, it is essential that your DNA/RNA samples pass appropriate quality assurance metrics before proceeding to NGS.