Highly efficient adapter ligation is the key to success for most NGS library preps. Robust, unbiased ligation is necessary to minimize loss in overall coverage during sequencing. Only “doubly-ligated” target DNA fragments (i.e. those containing adapters ligated at both the 5’ and 3’ ends) can serve as templates for Illumina sequencing, so maximizing the yield of doubly-ligated product is essential for maximizing efficiency of library production.
Increasing ligation efficiency maximizes library diversity in libraries constructed with both low and high amounts of sample input. Highly diverse libraries require less amplification for sequencing, resulting in lower duplication rates and improved coverage.
The scientists at Bioo Scientific have carefully optimized the ligation reaction step in NGS library prep to allow for the construction of the most diverse libraries possible with both low and high amounts of sample input.
A comparison of the NEXTflex Rapid DNA-Seq Kit to a competitor’s kit by analysis of proportions of ligation products. Red portions of the graph indicate non-ligated product proportion; yellow, singly-ligated; green, the desired doubly-ligated amplicon.
All of the NEXTflex Library Prep Kits incorporate Bioo Scientific’s proprietary Enhanced Adapter Ligation Technology into the adapter ligation reactions to improve library diversity.
Other tips for increasing ligation efficiency:
- Do not heat Illumina-compatible adapters above room temperature. They are constructed from two oligos which have been annealed together. Heating can cause the adapter to denature.
- Use the recommended adapter concentration dilutions for various input amounts. Using an excessive amount of adapters can result in an undesired increase in adapter dimers which will generate clusters that can be sequenced, and will reduce the total proportion of desired sequence obtained from a run.
Contact us at firstname.lastname@example.org if you have any questions about how improved ligation efficiency can improve your NGS analysis.