With so many methods for DNA size selection, it can be confusing to determine which is best for your project. Fortunately, academic researchers have evaluated many DNA size selection methods for a range of applications, and their conclusions provide some useful insight for the community.
Based on the original protocol for restriction site associated DNA (RAD) sequencing, double-digest RAD-seq incorporates an extra restriction enzyme step for use in massively parallel genotyping. The protocol is frequently used for organisms that don’t have a reference genome, allowing scientists to compare huge numbers of DNA variants in hundreds or even thousands of samples quickly and affordably.
The Harvard team that developed the ddRAD-seq method required incredibly precise sizing to make the process work, so they evaluated several size-selection options. They ultimately determined that manual gels did not deliver the necessary precision, noting that results were susceptible to operator variability. “Careful practitioners can achieve roughly 50% of the precision and repeatability of automated DNA size selection,” the scientists reported in their PLoS One publication of the method. “In contrast to automated size-selected samples, gel excision samples did not appear to saturate in the range of coverage observed.”
In another publication, scientists compared size-selection tools for a microRNA discovery pipeline with broader application among small RNA studies. Size selection for these elements is particularly challenging because the lengths are quite similar to the lengths of adapter-dimers and other unwanted artifacts. The team evaluated Novex TBE PAGE gels, the Pippin Prep automated DNA sizing platform from Sage Science, and AMPure XP beads, noting that their goal was to assess strengths and weaknesses of each option rather than to single out one option as the best.
Prior to sequencing, they found that both Novex and Pippin sizing led to a sharp peak at the expected microRNA size, while the beads included other size ranges. Notably, Pippin-prepared libraries “contained more than 50 times more product after purification, as compared to the Novex gel method,” the scientists report.
The libraries were then pooled and sequenced, resulting in a significant difference in total reads generated. Novex libraries produced an average of 8.8 million reads per sample, AMPure beads led to 9.1 million reads, and Pippin libraries generated 11.8 million. A control sample that had not been size-selected produced 8.5 million reads. The scientists also found that the methods yielded differences in the number of miRNAs identified, with an average of 372 from the control (non-sized) samples, 370 for AMPure, 415 for Novex, and 424 for Pippin.
The scientists identified certain strengths for each option. Novex gels were more specific for miRNAs than any other method; Pippin sizing was quite specific and produced the most total reads and distinct miRNAs; and AMPure beads were consistent and high-throughput, according to the paper.
Many types of experiments involve samples with very small volumes of DNA. In this publication, a team using metagenomics to study viral DNA in ocean water needed to reduce bias associated with amplification in order to produce enough sample for study. They focused on optimizing sample preparation methods, which included an evaluation of size-selection approaches. Their findings are useful for any workflow where low-input DNA is a concern.
The sizing comparison, which was undertaken to reduce size-related bias during amplification, included manual gels, Solid Phase Reversible Immobilization (SPRI) beads from Beckman Coulter Genomics, and the Pippin Prep. The scientists found that SPRI had low risk of contamination and was high-throughput, but it performed least efficiently in DNA recovery, yielding about half of the fractions collected by the other methods.
“Of the three sizing fractionation methods tested for target recovery efficiency (fraction recovered DNA in target 400–600 bp size range), throughput (ease of applicability to numerous samples simultaneously), and risk of cross-sample contamination,” the scientists report, “Pippin Prep, an automated optical electrophoretic system that does not require gel extraction, was the most efficient and reproducible (94–96% of input DNA), with the tightest, most specific sizing.”
Image from Tony Webster.
DNA size selection is a necessary but thankless task. Manual DNA sizing conjures images of postdocs slaving for hours over gels, trying to cut out specific bands with improbable accuracy. Automated DNA sizing, while vastly improving the precision and reproducibility of size selection, has historically been too low-throughput for many NGS labs or too imprecise […]