Miniaturization technologies for cost effective AmpliSeq library preparation for next generation sequencing

Purpose: AmpliSeq technology, the target enrichment method of next generation sequencing (NGS), enables quick and easy detection of the genomic hot spot region frequently mutated in species. Since the cost of next-generation sequencing has decreased, library preparation cost accounts for a more significant proportion of the total cost. If AmpliSeq library can be prepared at a lower cost, large-scale precision oncology can be more easily carried out. Furthermore, this technology can be widely applied not only to medical research, but also to polymorphism detection in biology. This study aimed to reduce the cost of AmpliSeq library preparation by adopting miniaturization technology. Methods: We used approximately 10 ng of genomic DNA for ultra-multiplex PCR of 384, 768, 1152, 1920, and 3072 amplicons. Multiplex PCR was performed in a total volume of 1.6, 2.0, and 2.4μL, using a nano-litre liquid handler, for library preparation. Results: The success rate of library construction decreased with decrease in total multiplex PCR reaction volume. Using 1.6-, 2.0-, and 2.4-μL reactions, success rate of ultra-multiplex PCR was 25 %, 95 %, and 100 %. We could stably create libraries of the correct amplicon size, with the amplicon number of approximately 1500 or less. As a result of NGS, uniformity of PCR amplification and read length of quality-checked libraries were hardly affected by the number of amplicons. Conclusion: Here, we present that minimum volume for a stable reaction was 2.4 μL, and maximum amplicons obtained were approximately 1500. The protocol saved 86.8 % in reagents, and reduced handling time by 85 %, compared to that in the manual protocol. Therefore, miniaturization technologies could reduce the cost of AmpliSeq library preparation through minimization of reagents.