IntroductionHigh-throughput genomic analysis is an indispensable and important tool in life science research, and this technology plays a vital role in promoting scientific research progress. In order to meet the urgent need for efficient and accurate preparation methods in the scientific research field, seqWell's Express Plex? Library Kit provides a solution specifically for high-throughput preparation of plasmid and amplicon libraries. In the traditional manual library preparation method, it takes up to 90 minutes to complete the preparation of 96 samples, of which 30 minutes are occupied by manual work. This method is not only inefficient, but also prone to errors due to human factors. To improve this situation, this article compared two library preparation methods, one using the seqWell ExpressPlex? Library Kit on an Opentrons Flex? pipetting workstation, and the other using manual library preparation. By combining the Flex Pipetting Station with the seqWell ExpressPlex? Library Kit, we can significantly reduce library preparation time and increase sample throughput. This automated solution not only improves preparation efficiency, but also reduces the risk of human error, providing a more reliable and efficient tool for life science research. Materials and Methods ExpressPlex workflow: According to the Express Plex workflow, we performed library preparation on a Flex automated pipetting workstation. During the experiment, we input 16 ng of pUC19, then added all components to the reaction plate, mixed at 3000 rpm for 90 seconds, and then performed subsequent library preparation and mixing steps. The final library was sequenced at 2X150 bp on a NextSeq 2000 sequencer.

Figure 1. A) Express Plex workflow includes adding index and DNA to the reaction plate (already containing enzyme and other mixtures), followed by amplification, mixing and MAGwise magnetic bead purification; B) Flex workstation includes tip boxes, modules, and transfer plates Grippers and other accessories; C) Flex workstation plate setup for library mixing of 4 reaction plates after amplification.

Results

Figure 2. A) The results show that the coefficient of variation (CV) of the library data in the 96-well plate is 12.7%; B) 95% of the library data passed the data preprocessing and is high-quality data, and more than 90% of the library quality scores reached Q30; C) Libraries prepared with Flex are more concentrated than those prepared manually.

Figure 3. Using staggered start times, 8 independent 96 mix 1 libraries can be prepared in 3 hours on Opentrons FlexTM. After setting up the first reaction plate on the Flex, 768 libraries can be prepared within 24 hours.

Conclusion The 96 pUC19 plasmid sample libraries prepared on the Flex pipetting platform showed highly uniform quality, fully proving the excellent performance of the Flex platform in automated library preparation. Compared to manual preparation, automated library preparation is not only more stable but also provides higher library concentration. In addition, more than 95% of reads successfully passed the sequencer quality inspection PF, which further verified the efficiency and accuracy of the automated preparation process. The Flex Pipetting Station supports up to 384 samples in a single run, greatly increasing sample processing efficiency. With proper timing, we can even process up to 768 samples in 24 hours, meeting the needs of large-scale, high-throughput genomic analyses. By automating the ExpressPlex? Library Prep Kit workflow on the Flex platform, we automate the entire process from extracted plasmid/amplicon DNA to data generation. This solution not only simplifies the operating steps and reduces human errors, but also significantly improves experimental efficiency and data quality, injecting new impetus into the development of the field of life science research.