Why Is It Important To Run Your NGS Gels Consistently?
Size Selection via Gel Electrophoresis
Whether you are using NGS for whole genome sequencing, SNP variant analysis, HLA typing, HLA matching, or even transcriptome or miRNA analysis by RNA-seq, size selection is an extremely important consideration for optimum results. Precise size selection can increase sequencing efficiency, save money and improve genome assemblies, as well as permitting the analysis of very low-input samples.
Many NGS library preparation protocols use agarose gels for size selection of genomic DNA, RNA or final libraries. Gel electrophoresis is a simple size selection technique and is very effective when carried out properly. Unfortunately, the simplicity of gel electrophoresis and it’s widespread use in other applications (e.g. crude visualization of PCR products) means that people often overlook the fact that it must be carried out very carefully for size selection. In this article, we provide our tops tips for consistent size selection by gel electrophoresis.
Four Top Tips for Success
1. Choosing the Agarose
Not all brands of agarose are the same! You can buy the basic and cheap agarose or the more expensive (low melting point and high-resolution) variants. Each of these is suitable for a particular technique and using a different product for each of your size selection gels will introduce differences in the pool of nucleic acids selected. Many protocols specify a particular product, so choose this product and do exactly what the protocol says. Most importantly, make sure you use the same agarose for all your experiments. This may mean buying agarose in bulk to ensure that you always have the right product to hand.
2. Making the Gel
It is important that you prepare your gels using a standard operating procedure, as you need to use gels with exactly the same strength in each run. Weighing everything carefully is key and it is worth weighing the final agarose: buffer mix before and after boiling to replenish any water that boils off during heating. This evaporation can change the concentration by a few percent, and it can impact the success of your size selection. Most labs use either TBE or TAE buffers. TBE is less likely to overheat, although TAE may be the best choice for NGS gels as the borate in the TBE can inhibit downstream reactions. There are also other buffers to choose from, and you can read about these in one of our other articles.
3. Running the Gel
Always use the same conditions for running the gel. Time, voltage and temperature are the biggest factors in determining what size products you are likely to cut out at the end of your experiment.
4. Removing Your Samples from the Gel
The easiest way to get your DNA out of the gel is to excise the correct band and extract the DNA using a gel extraction kit. However, simply using a scalpel to excise the gel piece is likely to produce variable results as it is difficult to cut the gel in exactly the same way every time. There are several suppliers of disposable gel cutting tips. These fit on the end of a pipette and result in identical gel cuts every time. Of course if you don’t follow steps 1, 2 & 3 carefully, then your results will vary every time anyway!
When Your Software Does Not Compute
Small variations can lead to significant differences in insert sizes. If the software you are using does not compute insert size from the sequence reads, then things like structural variation analysis or peak-shifting can be adversely affected.
If It Goes Horribly Wrong…Don’t Panic!
One very useful tip is to cut out multiple bands. If something goes horribly wrong downstream you still have a sequencing library to go back to! However, by following the above tips, you should get the same size NGS library each time you prepare a new set of samples.
Originally published in 2013. Updated and republished in June 2017.