In many NGS workflows, you want more than just a single slice of your DNA sample — but most size selection methods make it difficult to salvage multiple bands or size ranges of DNA. This is especially important when dealing with precious samples or with certain applications where it’s useful to have libraries with multiple insert sizes, such as transcriptome or mate-pair sequencing.
Sage Science developed the SageELF automated whole-sample fractionation system to meet this need. SageELF is an electrophoresis-based instrument that separates DNA by size and collects the whole sample in 12 size-specific fractions. It can resolve short or large DNA fragments.
Scientists are using the system for a number of NGS applications, including some that wouldn’t be possible without SageELF. At The Genome Analysis Centre, researchers published a new method for mate-pair sequencing based on the instrument. The protocol saves time and money and requires less input DNA than other mate-pair approaches by ensuring the insert size of NGS libraries. “Using the SageELF streamlines the library construction process, allowing [long mate-pair] libraries >10 kb to be constructed in under 2 days with <10 µg input material,” the scientists report in the paper. “For many genome projects, multiple insert size LMP libraries are required, and the ability to construct up to 12 discretely sized libraries for a combined reagent cost of $1270 compared with the reagent cost of $715 for a single insert size LMP library highlights the potential cost savings.” The authors report that improved accuracy with insert sizes leads to more straightforward scaffolding and assembly downstream.
The same TGAC team also deploys SageELF for analysis of copy number variation. The instrument is used to fractionate PCR products so they can sequence the largest fraction to find the most copies in a sample.
The SageELF has also proven important for isoform sequencing with PacBio and is included in this protocol. The company recommends using SageELF to fractionate cDNAs into four pools, with an optional step to remove smaller fragments prior to sequencing if appropriate. In this poster, scientists from PacBio and the University of Washington present data showing the utility of SageELF in a gene expression study of human cell types.
Find out more about the SageELF system on the Sage Science website.
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. Double-Digest RAD-Seq Based on the original protocol for restriction site […]