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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\u00ae Library Quantification Kit, pooled in 8x or 16x format into single samples, and then analyzed in the Yili\u00ae MiSeq Run on a 2x75 flow cell. Sequencing data were extracted and demultiplexed based on the Yili\u00ae DNA\/RNA Unique Dual (UD) Index Adapter barcode sequence.<\/p>\n\n\n\n
Workflow Layout The layout of the OT-2 platform includes modules, laboratory software, and Ilimina\u00ae 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.<\/p>\n\n\n\n
For PCR amplification of DNA preparation samples, Yili\u00ae 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\u2122 dsDNA High Sensitivity Detection Kit (Thermo Fisher Scientific, Cat. No. Q33120) on a Qubit\u00ae 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.<\/p>\n\n\n\n
Small Genome Sequencing of DNA Prep Libraries Labeled with 96 Samples\u00ae DNA Prep Kit (M). E. coli unmethylated genomic DNA (Zymogen, Cat. No. No. 20018705) was prepared. Sample (n=16,100ng)<\/p>\n\n\n\n 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.<\/p>\n\n\n\n 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 \u200b\u200bof the 8x and 16x\u00ae 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\u00ae 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).<\/p>\n\n\n\n Conclusion We demonstrated its high performance and performance. Automate the labeling process using the Illumina\u00ae 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<\/p>\n","protected":false},"excerpt":{"rendered":" Authors: Matthew Akana, Dr. Kinneri Watson, Dr. Molayo […]<\/p>\n","protected":false},"author":5,"featured_media":3039,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"inline_featured_image":false,"_monsterinsights_skip_tracking":false,"_monsterinsights_sitenote_active":false,"_monsterinsights_sitenote_note":"","_monsterinsights_sitenote_category":0,"footnotes":""},"categories":[],"tags":[],"class_list":["post-3037","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry"],"yoast_head":"\n![\"\u7532\u677f\u5e03\u5c40\u914d\u5907\u6a21\u5757\u3001\u5b9e\u9a8c\u5ba4\u5668\u76bf\u548cIllumina](\"https:\/\/opentrons.com.cn\/wp-content\/uploads\/2024\/06\/image-94.png\")
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