BioSpotlights

Enriching your RNA-seq efforts

When it comes to any next-generation sequencing experiment, scientists want to maximize the number of informative reads. In the case of high-throughput RNA sequencing (RNA-seq), this means eliminating those RNA species that are not of interest for the experiment. The trouble is that, in the RNA world, those RNAs that are not of direct interest are often the ones that are most abundant. A number of techniques have been developed to enrich for specific classes of RNA prior to RNA-seq, including methods based on the presence of poly-A tails, size selection, or hybridization to specific DNA oligonucleotides. While these approaches have proven effective in many cases, problems can still arise. Take for instance the case of Drosophila small RNA sequencing (miRNA, piRNA, siRNA). Here, the RNAs of interest are 20–30 nucleotides in length, but so too is the highly abundant class of 2S rRNA, making small RNA enrichment in this species a particular challenge. But in a Benchmark article in this issue of BioTechniques, Michelle Wickersheim and Justin Blumenstiel from the University of Kansas present a clever solution to enriching for small RNAs—one that does not require additional hybridization or size selection techniques. Wickersheim and Blumenstiel hypothesized that 2S rRNA could be most effectively depleted during the ligation and cDNA synthesis steps of sequencing library construction by not allowing these RNAs to take part in the reactions. The authors accomplished this depletion using terminator blocking oligonucleotides specific for 2S rRNAs that annealed to the templates prior to the 5′ adapter ligation step. In this way, only small RNAs of interest should be reverse transcribed, barcoded, and subsequently sequenced. When Wickersheim and Blumenstiel tested their terminator oligo blocking methodology, they found less than 0.1% of total reads obtained following depletion using blocking oligos were from 2S rRNA. Although the method was demonstrated for Drosophila small RNAs, this approach will likely find favor with all scientists interested in enriching their target small RNA populations.

See “ Terminator oligo blocking efficiently eliminates rRNA from Drosophila small RNA sequencing libraries ”

The simple life

Simplicity is wonderful. If your need five steps to accomplish your goal, that is fine, but if it can be done in two, so much the better. Working at the bench is no different—a protocol with two steps versus five that works well saves time and money and is obviously worth testing out. With this in mind, in this issue of BioTechniques Liang et al. detail a simplified, one-pot strategy for recombinant cloning. Gateway cloning is a recombination-based cloning methodology that uses bacteriophage lambda to move sequences containing compatible recombination sites between different vectors. In the Gateway approach, entry vectors and destination (expression) vectors are usually generated to move DNA segments. The simplicity of this methodology lies in the ability to generate new expression vectors rapidly without the need for traditional ligase based cloning. Although straightforward, recombination with the Gateway system still requires the use of two enzymes (BP and LR) in two separate reactions. But Liang et al. realized that the difference between the BP and LR reactions was the presence of lambda excisionase and that there might be a level of excisionase activity where both reactions would proceed at a similar rate. If this were the case, a single BP/LR reaction would be possible. The authors set out to test their idea by using a DNA fragment harboring a reporter gene (in this case lacZ) and testing different ratios of LR/BP. Their results clearly showed that one pot recombination cloning was possible with the Gateway system, with elevated LR/BP ratios resulting in more expression clones while reciprocal ratios resulted in more entry clones. In the end, this simple yet powerful technique for recombinat cloning will not only decrease time spent at the bench, but also lower the cost of this type of cloning in general.

BP/LR reactions in the Gateway recombination system.

See “ Single step BP/LR combined Gateway reactions ”