A modified protocol with less clean-up steps increased efficiency and product yield of sequencing library preparation.

Library preparation is an essential step for the next-generation sequencing, such as whole-genome sequencing, reduced-representation genome sequencing, exome sequencing and transcriptome sequencing. The library preparation often involves many steps, including DNA fragmentation, end repair, ligation and amplification. Each step involves different enzymes and buffer systems, so many washing steps are implemented in between to clean-up the enzymes and solutes from the previous step. Those extra washing steps not only are tedious and costly, but more importantly may introduce cross-contamination and reduce the final library yield. Here, we modified the common protocol of Illumina library prep to reduce the washing steps by deactivating the enzymes with high temperature. The modified protocol has two less washing steps than the original one, which can save more than 40 min of hands-on time and reduce potential risk of cross-contamination. We compared our protocol with the original one by constructing libraries using 200 ng DNA of Tetraodon nigroviridis. The results showed that libraries prepared with the modified protocol had higher yields than that using the original protocol (53.4 ± 16.8 ng/ml vs. 8 ± 0.7 ng/ml), whereas the coverage and PCR duplication rate were similar. Furthermore, we eliminated the very first washing step after DNA shearing to preserve short DNA fragments, which increased proportion of fragments less than 100 bp DNA from 0.82 to 2.99%. In conclusion, using the modified protocols not only can save time and money, but also can generate higher yield and keep more short DNA fragments. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03168-5. © King Abdulaziz City for Science and Technology 2022.