Automatically prepare small genome DNA libraries using the ?DNA Prep Kit on the OT-2
Authors: Matthew Akana, Dr. Kinneri Watson, Dr. Molayo Adeibi.
Introducing next-generation sequencing (NGS) workflows including DNA library preparation using commercially available software and cost-effective DNA preparation kits. It is helpful to optimize this workflow by using the automated OT-2 protocol to increase DNA library preparation efficiency. Here we describe the efficiency of Yili? DNA. The preparation kit (Illumina, CatNo. 20018705) automates the labeling workflow on the OT-2, robotic liquid handling platform. Automating this process using the OT-2's high-performance capabilities provides a fast and robust workflow that generates uniform DNA-prepared libraries for genome sequencing.
Overview of the Illumination? DNA Prep Kit and OutoCenter OT-2 Platform The Illumination? patented NGS labeling workflow enables on-bead labeling of linked transposomes utilizing beads. The chemistry of the bead-chain transposome is more efficient for DNA fragmentation and DNA end repair compared to previous in-solution labeling workflows. The DNA fragments are tagged using dual-index adapters, a unique barcode assigned to each DNA fragment. DNA Prep samples (n=8) were individually quantified, normalized to a 4nM library, pooled into a single multiplex (8x or 16x) sample, and then genomically performed on a 2x75 flow cell on an Illumina? MiSeq Sequencing. Our data show low variability across all data, DNA preparation samples assayed before and after PNA amplification; barcode balancing, and uniform and high-coverage sequencing alignments. Studies of reference genomes demonstrate that automated labeling using the OT-2 protocol provides a high-quality DNA-prepared library for genome sequencing.
Schematic of the OT-2 workflow protocol for DNA and IDT? versus DNA/RNA UD Index Set A for DNA and IDT?. 20027213) During incubation a process called labeling occurs on OT-2, as shown in the figure (Figure 1). A labeling wash step was performed to remove residual DNA and adapters. Next up is the OT-2 method for DNA amplification with an on-deck thermal cycler with programmable lid and block temperatures.
Figure 1. Illumina DNA preparation workflow. Dotted boxes represent steps of the automated Illumina DNA preparation workflow on OT-2
Following the OT-2 protocol, perform post-amplification using AMPure (Beckman Coulter, Cat. No. A63881) to detect DNA according to manufacturer recommendations. Prep samples were quantified, normalized to 4 nM (qPCR assay for library quantification using the NEBNext? Library Quantification Kit, pooled in 8x or 16x format into single samples, and then analyzed in the Yili? MiSeq Run on a 2x75 flow cell. Sequencing data were extracted and demultiplexed based on the Yili? DNA/RNA Unique Dual (UD) Index Adapter barcode sequence.
Workflow Layout The layout of the OT-2 platform includes modules, laboratory software, and Ilimina? DNA preparation reagents, as detailed (Figure 2). While the workflow includes automated pipetting on the OT-2, manual intervention is required to move the sample plate between the thermal cycler pen and magnet on the deck, and to reset the pipette tip rack during the protocol.
For PCR amplification of DNA preparation samples, Yili? DNA Preparation Kit was used to construct a Lambda DNA library on OT-2 (n=8,100 ng). Quantify pre- and post-PCR concentrations (ng/uL) of 5 cycles of PCR amplification, and DNA concentration using the Quant-iT? dsDNA High Sensitivity Detection Kit (Thermo Fisher Scientific, Cat. No. Q33120) on a Qubit? Determine the coefficient of variation, which is the standard deviation divided by the mean for the entire sample. The average pre-PCR concentration of Lambda DNA samples was 2.92 and the coefficient of variation (CV) was 10.4%, and the average post-PCR concentration after 5 cycles was 28.23 and the CV was 10.7%, see (Table 1). These results indicate that the optimized OT-2 protocol can produce a high-quality and reliable NGS DNA library preparation.
Small Genome Sequencing of DNA Prep Libraries Labeled with 96 Samples? DNA Prep Kit (M). E. coli unmethylated genomic DNA (Zymogen, Cat. No. No. 20018705) was prepared. Sample (n=16,100ng)
Figure 2. OT-2 deck layout equipped with modules, labware, and Illumina DNA preparation reagents.
This workflow has automated pipetting capabilities on the OT-2 and requires manual operations to move the sample plate (1) and magnetic block (2) on the deck, as well as reset the tip rack during the run. The deck layout includes 1x NEST 12-well reservoir, 1x 96-well aluminum block, 1x 200ul PCR plate on an Eppendorf thermal cycler, and a 1xNEST 0.1 mL PCR plate temperature module (3). Modules include a magnetic module, temperature module, thermal cycler module, and P20 and P300 multichannel pipettes.
Table 1. Low variability between library construction on Opentrons OT-2.This table shows the yield of each sample after Illumina tag wash over 5 cycles of PCR Amplification and AMPure CleanupTable 2. Sequencing batch specifications for small genomes showing low variability. Acomparison of 3 different batches prepared on the OT-2 using the Illumina DNA preparation workflow (1). Yields from different sample types are consistent (2) and individual sample barcodes show a uniform representation (3). The average sample genome coverage approximates the expected coverage calculated from the Illumina coverage calculator. Figure 3. Highly consistent sequencing coverage across small genomes. Coverage map from genome shows ~7800x of Lambda genome covering 48kb
Two batches, second batch, E. . E. coli DNA samples (n = 8,100 ng) were processed on 2 separate columns on the OT-2 to account for possible intra-column variability. E. coli DNA (n=16,100 ng) and Lambda DNA (n=8,100 ng) were amplified, normalized, and pooled into a single multiplexed (8x or 16x) sample for sequencing. Sequencing results of three DNA preparation batches were compared to determine sample yield, barcoding, balance, and coverage. As shown in (Table 2), uniform yields were observed for the DNA preparation library. The sample yield of E was calculated. The E. coli content in Laboratory 8 was 9.75 and 3.79. 16x respectively; the CV values ??of the 8x and 16x? adapter barcodes were 8.77% and 6.2% respectively, showing that the balance of the 8x and 16x samples was accurate 8x The average of 11.2%-12.31%, compared with the expected 12.5%. For 16-plex, the expected value of 6% calculated based on the Yili? reference is 6.2%. Average genome coverage of DNA prepared E. The 8x for the E. coli sample was 150x and the 16x was 45x. Additionally, the coverage map of the Lambda 48kb genome showed ~7800x, indicating a higher level of aligned sequence reads (Figure 3). Overall, this shows that DNA Prep libraries constructed on OT-2 for sequencing show consistency compared to reference calculations (Figure 4).
Figure 4. Unified sample barcode representation. This pie chart shows the percentage of even sample barcode balance in a sequencing run by overall reads
Conclusion We demonstrated its high performance and performance. Automate the labeling process using the Illumina? DNA Preparation Kit on the Vision Center OT-2. We demonstrate that DNA Prep samples display low variability, barcoding balance, and high coverage of sequence alignments. Automating this comprehensive NGS workflow on the OT-2 minimizes risk during DNA library preparation while ensuring genomic DNA library preparation quality sequencing
all · 2025Year18Moon19Day
