Evaluating Library Creation Efficiency
Sample preparation for the majority of NGS platforms follows a similar protocol. Genomic DNA is sheared to an appropriate size, end repaired, and in most cases A-tailed. Adapters are ligated and the final product is purified or size selected. If needed, the library will be subjected to limited amplification by PCR. Because shearing results in DNA fragments with overhangs, ends must be repaired prior to the addition of 3´ A tails or the ligation of blunt-end adapters. A-tailing of target DNA in combination with ligation to T-tailed adapters is designed to prevent the formation of chimeras and concatemers formed by self-ligation of both target DNA and adapters.
Although kits that perform end repair and tailing in a single tube are simple to use and fast, they are generally not optimized for A-tailing and will add both 3´ adenine and guanine residues at approximately equal rates. The result of inefficient tailing is a mixture of DNA with blunt, A-tailed, and G-tailed ends, and consequently, inefficient ligation of adapters and low complexity libraries.
Lucigen developed a molecular assay for end repair and tailing that allows the quantization of the efficiency of tailing and ligation. In the absence of tailing, a ladder of concatemers will be visible in the assay indicating blunt ligation. Target DNA consists of a double-strand synthetic 90 mer that must be end repaired and kinased before it is ligatable. Adapters were designed having either a T-tail or a C-tail to assess ligation to A-tailed target or G-tailed target, respectively.
Target DNA that is end repaired, but not tailed, will form concatemers when ligated. A target that is G-tailed will ligate to the C-tailed adapter. Before end repair, the target DNA will not self-ligate to form concatemers. After end repair, but prior to A-tailing, the target DNA will self-ligate and form a ladder of concatemers (Figure 1). T-tailed and C-tailed adapters will not self-ligate and will ligate only to target DNA with A- or G- overhangs, respectively.
To benchmark the optimized NxSeq™ technology, two leading NGS library kits representing two different sequencing platforms were compared using the previously described assay. In Figure 2, an ethidium bromide-stained gel demonstrates the efficiency of A-tailing and subsequent ligation to T- or C-tailed adapters for the three systems.
The expected products are denoted on the left side of the gel, with the blue bar representing the 90 mer target and the red bars representing ligated adapter.
If A-tailing is occurring at a higher rate than G-tailing, the intensity of the desired band in the “T-adapter” lanes should be stronger. Examination of the existing NGS library kits reveals equal amounts of A- and G-tailing, along with notable presence of bands that can only exist by the formation of concatemers and chimeras. In contrast, the Lucigen library exhibits the high efficiency of a T-tailed ligated adapter with essentially no side products (concatemers or C tails).