There are several methods for size-selecting DNA fragments prior to sequencing. How do you choose which is best? Here’s a look at various options, plus considerations to help you determine when to use each one.
Virtually every student in a biology lab knows how to prepare and cut a manual gel—but their ubiquity doesn’t mean they’re ideal for all scenarios. Because they require so much hands-on time, these gels make sense for labs that seldom prepare NGS libraries, where sizing is only needed once in a while. They’re also a great fit when you’re not sure where your sample is—that is, you have no idea how big the DNA fragments might be. With manual gels, it’s straightforward to visualize and extract your DNA. Automated techniques are often dye-free to prevent DNA damage, so they require some up-front knowledge of approximate fragment size.
Bead-based protocols have been incredibly helpful for labs interested in automating very high-throughput, production-scale sequencing workflows. Beads are quite useful in cleanup steps for NGS libraries. For size selection, beads deliver a fairly broad distribution of size fragments, so they should not be used when more precise sizing is needed. They also lose more of a sample than other methods, so they’re most useful in experiments where DNA is abundant.
If you’re working with large DNA—anything greater than 10 kb—then you’ll need a pulsed-field gel.1 Long pieces of DNA get clogged up going through gels, so the pulsed-field approach gets around this by applying electrical fields in different directions, alternating the voltage to prevent the fragments from getting stuck together. Pulsed-field gel electrophoresis (PFGE) lets you resolve ultra-long DNA, even megabases in length. Many PFGE platforms are used for analysis rather than for size selection, helping scientists determine how long the DNA pieces are for a variety of applications.
Automated DNA Size Selection
Automated size selection using precast, disposable gel cassettes delivers the benefits of a gel (more precise sizing with better yield) with the reduced hands-on time and higher throughput of beads. Platforms like those designed by Sage Science can handle small or large DNA, making them handy when building libraries for short-read or long-read sequencers. Studies have shown that automated size selection produces more reproducible results than manual gels, and that users can get higher-quality data using less DNA.2-4
- Cantor CR, Smith CL, Mathew MK. (1988) Pulsed-field gel electrophoresis of very large DNA molecules. Annu Rev Biophys Biophys Chem. 17:287–304.
- Quail MA, Gu Y, Swerdlow H, Mayho M. (2012) Evaluation and optimisation of preparative semi-automated electrophoresis systems for Illumina library preparation. Electrophoresis. Dec;33(23):3521–8.
- Duhaime MB, Deng L, Poulos BT, Sullivan MB. (2012) Towards quantitative metagenomics of wild viruses and other ultra-low concentration DNA samples: a rigorous assessment and optimization of the linker amplification method. Environ Microbiol. 14(9):2526–37.
- Lopez JP, Diallo A, Cruceanu C, Fiori LM, Laboissiere S, Guillet I, et al. (2015) Biomarker discovery: quantification of microRNAs and other small non-coding RNAs using next generation sequencing. BMC Med Genomics. 8:35.