Summary The Swift2S? Turbo DNA Library Kit is automated with OT-2 for rapid and robust preparation of high-quality libraries for next-generation sequencing. .Libraries with 100 and 1 ng input were prepared from mixtures of bacteria with varying GC content, including: Staphylococcus aureus, Escherichia coli, and Streptomyces avermida. .The performance of hand-prepared libraries and openteronOT-2 is comparable in terms of scale and throughput. Metrics such as complexity, coverage, and relative representation were also observed for similar sequencing.

Introduction Next-generation sequencing (NGS) library preparation requires multiple steps for fragmentation, polishing, adenylation, ligation, and PCR, requiring 2 to 5 master mix additions, incubations, and SPRI cleanup. Laboratory automation can reduce the hands-on time of a technique while increasing the reliability of experiments due to human error and contamination. Opt-2 is an affordable liquid handling platform, starting from $5,000, that enables automation of complex library preparation. Automation and implementation using the powerful fluidics of the Swift2S? Turbo DNA Library Kit (1) ensures fast and consistent performance for next-generation sequencing. A two- to three-hour Turbocharger protocol allowing varying input amounts of DNA (1 to . 250 ng), volumes, and sample types has been demonstrated. Featuring a simulated bacterial community, the Swift2S? Turbo DNA Library Kit with OT-2 is a fast and ideal library preparation solution for whole-genome sequencing, targeted exomes, genotyping, and metagenomics. Learning and other applications.

Methods and Materials A library was prepared using the genomic DNA of three bacteria with different GC contents, namely Staphylococcus aureus (ATCC 25923D-5), Escherichia coli (ATCC 700926) and Streptomyces (ATCC 31267), mixed at the same molar concentration of DNA. . Libraries are hand-prepared by experienced Swift developers with 6 or 8 DNA Technology replicates, with 100 ng and 1 ng input per library ready. 8 technical replicates. Both inputs were also prepared on OT-2. The fragment master mix used in the 100 ng input sample prepared on OT-2 included RegentDE to slow down the fragmentation reaction (2). Add 5 ul of reagent DE to each reaction without adding reagent K2, increase the fragmentation time to 15 minutes, and prepare a total insert size of 350 bp. Use the PCR method to combine each indicator with 100 ng and 1 ng for 5 and 11 cycles respectively. 8. Randomly select libraries for sequencing, including input from OT-2 and manual sequencing. All sequencing is on Illumina MiSeq standard V2 PEx150. conduct. The rotor temperature and magnetic modules are used for active cooling of enzymes and magnetic bead cleaning protocols on the OT-2. For comparability analyses, reads were normalized to 1.9 million reads per sample.

Swift2S? Turbo DNA Library Kit (left). All these experiments used the thermometer module required for active cooling of the enzyme and the magnetic module required for bead cleaning. Thermal cycler modules can be added to the OT-2 deck for full automation. The Swift2S? Turbo protocol includes enzymatic shearing of input DNA, adapter ligation, indexing, or optional PCR. Use SPRI cleanup after ligation and PCR steps.

Results Along with the time savings of automation, Swift2S? prepared Turbo libraries on OT-2 are robust and comparable to manually generated libraries. To illustrate the unbiased performance of Swift2S? Turbo OT-2, the library input was 100 ng and a bacterial mixture of three strains with GC content (33 -71%) was used. The library was prepared by an experienced developer of both the Swift2S? Turbocharger and the Opentrons OT-2 reagent DE, used to slow down the fragmentation of enzymatic reactions in chambers for longer periods of time in automated liquid handling The process temperature (2) is added to the fragmentation master mix program. Thus, comparable to the library size, the yield was 85% compared to the manually prepared library on the OT-2 version (Figure 2). The CV of the library yield was 14% across 8 technical replicates prepared on OT-2.

Sequencing metrics are also comparable when prepared manually and on the OT-2. For example, 9% of reads mapped to the reference genome in the bacterial mixture, indicating no contamination or reduced infidelity in libraries prepared on OT-2 (Figure 3). Comparable percentages of duplicates observed in manual and automated preparations indicate that DNA is retained throughout the SPRI cleanup operations performed on OT-2 (Figure 3). Comparable performance and sequence representation were further demonstrated by consistent coverage and relative abundance between automated and manual library preparers (Figure 4).

Figure 3. Manually compiled library of comparable ranking indicators. Manually prepared libraries are shown in yellow, libraries prepared on the opentronOT-2 are shown in blue. The left and right panels show similar read volumes and percentages of duplicates aligned to the reference genome. These metrics indicate that no contamination or loss of input DNA affects library complexity.

Figure 4. The relative abundance and coverage of the hand-prepared library and the library on the opentronOT-2 were comparable. Libraries were normalized to have the same number of sequencing reads (1.9 million reads) for comparative analysis.

Conclusion Automated library preparation reduces the potential for human error and reduces hands-on time and reagents without sacrificing the quality of library preparation. This automated Swift2S? Turbo protocol on the OT-2 platform enables robust preparation of 8 or more libraries in 2 to 3 hours with minimal yield variation between technical replicates. .Reagent DE (2) is used for automatic settings at room temperature to slow down fragmentation. .Libraries prepared using Opentrons OT-2 were comparable and reproducible (CV=14%). .Similar sequencing metrics were observed for manually prepared libraries and libraries prepared on OT-2, with comparable complexity, bias, and coverage.