Sequence Preparation Fully automated library preparation of microbial and human DNA templates

Authors Red and Kinnery Watson

Summary The Swift 2S Turbo DNA Library Kit is a fully automated, hands-off, high-quality library for Illumina next-generation sequencing using the Center OT-2. Key findings. An 8-sequence prepared DNA library can be constructed in 3 hours. .Target insert sizes of 326bp and 330bp were analyzed on human genomic DNA. .Low PCR bias, indicating PCR repeat levels of 0.02% and 0.01% for microbial and human libraries, respectively. .High DNA coverage was maintained during the OT-2 run, with alignments described by similar percentages of reads: 95.4% for the microbial library and 96.4% for the human library. .Reliable and accurate performance of the Thermocycler Module compared to third-party thermal cyclers through similar yields of 5.1ng/µl to 7.36ng/µl with CVs of 13% and 14% respectively.

Introduction Next-generation sequencing (NGS) library preparation is the process of enzymatic sequencing of RNA or DNA fragments. The use of NGS library preparation is rapidly increasing, providing healthcare and life sciences researchers with a variety of workflows to enable faster , an efficient way to analyze and interpret large genomic data sets more easily and at higher cost (1). Swift 2S Turbo DNA Library Preparation

Supports molecular applications for library preparation on a variety of samples. This automation-friendly workflow is compressed, but completing the workflow manually will still result in pipetting errors and take several hours to complete. Here we illustrate the complete automation of Swift 2S Turbo library preparation for microbial as well as human genomic DNA. Complex library preparation can be fully automated using Vision Center OT-2, modules, laboratory software, and Omega Bio-Tek Labeled® pure NGS beads.

Materials and Methods A library of 350 bp insert size ~ 560 bp library was prepared using human genomic DNA (Coliella sp. NA12878) and Ecoli (ER2925) gDNA with GC content of 40 and 50%, respectively. Each opentronOT-2 run contains 7 replicates, and each run contains 5 or 10 PCR cycles, depending on the startup input. The ligation master mix used includes a 1:10 dilution of Reagent W4 to reduce adapter dimers. Each library was sequenced on an Illumina MiSeq Nano v2 2x250 kit. Optional rotor thermal cycler module and temperature module for active cooling of master mix, index and sample bead-based cleanup using Omega-Beo-Tek Mag-Bind® pure NGS beads (2). Together these modules allow complete automation of this composite workflow (Figure 1). OT-2 was run with the P300GEN2 8-channel pipette and the P50 GEN2 single-channel pipette with E. coli gDNA, and with the P300GEN2 8-channel pipette and the P20 GEN2 single-channel pipette with human gDNA. However, we recommend using P20.GEN2 single-channel pipettes with 3 tips per run (Figure 1). The total run time, including hands-on time and robot run time, was approximately 3 hours (Figure 2). Peak fragment sizes were determined using Agilent 2100 Bioanalyzer fastq files.

Analysis was performed using FASTQC V0.11.9 (3), BOWTIE2 (4), Qualimap-BAMQC (5) and BAM coverage V2.4.1.0 (6) in Galaxy (7) to determine coverage, insert size, reads Alignment to genome, GC content and repeat percentage. BWA aligner V1.1.4 for further analysis.

The resulting library yields are highly reproducible and comparable within the same run across samples and across species. Ecoli gDNA input is 100 ng, human gDNA input is 77 ng. . . The Ecoli library workflow consisted of 10 PCR cycles with an average concentration of 20.34ng/µl and a coefficient of variation (CV) of 7%. The human gDNA library workflow consisted of 5 PCR cycles with an average cycle of . The libraries were comparable with an average peak fragment size of 591 bp and a CV of 7%. The average peak fragment size of the human library was 569 bp and a CV of 4%. Yield and peak fragment size CVs showed consistency across wells and species (Figure 3). Sequencing metrics are also comparable across samples and species. The average insert size for the E. coli and human libraries was 326 and 330 bp, respectively, corresponding to the DNA target length incorporated into direct sequencing according to the reagent specifications. Both experiments showed that ~95% of reads mapped aligned to the reference genome sequence, with high DNA recovery and size selection consistently retained in every run, illustrated by the very low PCR repeats (Figure 4). . Consistent coverage in Ecoli samples (Figure S1) and human samples (Figure S2) showed identical performance and sequence representation. And the single nucleotide variant (SNV) rs6061194 further demonstrates this performance (Fig. S3). Finally, the performance of the central thermal cycler module is similar to that of the manual third-party thermal cycler (8), with commonalities in throughput and CV. The yields were 5.1ng/µland and 7.36ng/µland respectively. The accuracy and reliability of this end-to-end Opentrons library preparation system (Table 1).

Conclusion Opentrons OT-2 can deliver 8 or more high-quality libraries at a time in at least less than 3 hours, ultimately reducing the risk of manual pipetting errors and saving user time. .Reproducible and consistent sequencing metrics in yield, CV, insert size, alignment, PCR repeats and coverage of microbial and human libraries. .Consider the reliable and precise performance of the central thermal cycler module compared to third-party thermal cyclers when yield and CV are .

Figure 1. Swift 2S Turbo workflow and OpSwift 2STurbo DNA Library Kit© protocol. This layout includes the P20 GEN2 single-channel pipette and the P300GEN2 8-channel pipette. Module requirements include a view center thermal cycler module, a temperature module and a magnetics module. This labware requirement includes two NEST 0.1ml 96-well PCR plate full skirts, one NEST deep 12-well reservoir, three NEST 2ml screw-cap tubes and a center 24-well aluminum block.