Maximizing the efficiency of NGS library preparation is particularly crucial when working with limited sample quantities. Biological samples from patients, especially those like FFPE or cfDNA, may have limited amounts of DNA, making it challenging to use sufficient amount of DNA for NGS analysis. Therefore, using high-efficiency DNA library preparation kit for subsequent experiments such as Target Enrichment can be beneficial.
At Celemics, we provide customers with high-quality and high-efficiency kits to ensure optimal results. Additionally, we devote efforts to the development and optimization of experimental methods. As part of these efforts, this post aims to introduce a method for maximizing library yield by diluting adapters during library preparation when working with limited DNA input.
1. Results from Blood or Tissue Genomic DNA
Typically, high-quality genomic DNA which is extracted from blood or tissue samples and therefore can easily provide sufficient quantities for library preparation. Celemics’ library preparation kit recommends a minimum input of 50 ng, which will provide sufficient library amounts when followed accordingly. However, for further optimization, we have also compared yields by diluting adapters; the results showed that diluting adapters by 1/20 could provide higher yields compared to the standard methods.
2. Results from FFPE Genomic DNA
DNA from FFPE samples often presents limited quantities and with lower DNA quality. This obligates customers to proceed to constructing libraries with quantities below the recommended input. To address this issue, we have investigated whether the Celemics kit can provide adequate yields even with an input DNA amount as low as 25 ng and how yield changes with respect to series of adapter dilutions. The results demonstrated that diluting adapters by 1/20 resulted in approximately four times higher yield compared to previous methods.
3. Results from Cell-free DNA
Lastly, cell-free DNA may challenge the library preparation with even smaller quantities than FFPE samples. In order to determine the most optimized experimental condition, we have examined the efficiency changes in library preparation by diluting adapters with respect to changes in input DNA quantities of 5 ng and 20 ng. The findings revealed that, even with lower input quantities (as low as 5 ng), diluting adapters by 1/10 resulted in the highest efficiency.
In summary, these experiments confirm that high-efficiency libraries can be obtained with minimal input DNA when using Celemics’ library preparation kit. Furthermore, our experimental results have demonstrated that diluting the adapters can further enhance the efficiency, providing the most optimized results especially when working with samples of nucleic acids with limited quality and quantity.