Menu
2021年06月01日

Single chromosomal genome assemblies on the Sequel System with Circulomics high molecular weight DNA extraction for microbes

Author(s): Wong, J. and Heiner, C. and Kim, M. and Ferrao, H. and Wallace, V. J. and Eng, K. and Fedak, R. and Kilburn, D. and Ashby, M. and Baybayan, P. and Burke, J. M. and Liu, K. J.

Background: The Nanobind technology from Circulomics provides an elegant HMW DNA extraction solution for genome sequencing of Gram-positive and -negative microbes. Nanobind is a nanostructured magnetic disk that can be used for rapid extraction of high molecular weight (HMW) DNA from diverse sample types including cultured cells, blood, plant nuclei, and bacteria. Processing can be completed in <1 hour for most sample types and can be performed manually or automated with common instruments. Methods:We have validated several critical steps for generating high-quality microbial genome assemblies in a streamlined microbial multiplexing workflow. This new workflow enables high-volume, cost-effective sequencing of up to 16 microbes totaling 30 Mb in genome size on a single SMRT Cell 1M using a target shear size of 10 kb. We also evaluated this method on a pool of four “class 3” microbes that contain >7 kb repeats. Fragment size was increased to ~14 kb, with some fragments >30 kb. Results: Here we present a demonstration of these capabilities using isolates relevant to high-throughput sequencing applications, including common foodborne pathogens (Shigella, Listeria, Salmonella), and species often seen in hospital settings (Klebsiella, Staphylococcus). For nearly all microbes, including difficult-to-assemble class III microbes, we achieved complete de novo microbial assemblies of =5 chromosomal contigs with minimum quality scores of 40 (99.99% accuracy) using data from multiplexed SMRTbell libraries. Each library was sequenced on a single SMRT Cell 1M with the PacBio Sequel System and analyzed with streamlined SMRT Analysis assembly methods. Conclusions: We achieved high-quality, closed microbial genomes using a combination of Circulomics Nanobind extraction and PacBio SMRT Sequencing, along with a newly streamlined workflow that includes automated demultiplexing and push-button assembly.

Organization: PacBio
Year: 2018

View Conference Poster

咨询专家

如果您有疑问、需要查看订单状态或想要购买仪器,我们随时乐意提供帮助。

姓名(Required)
这个字段是用于验证目的,应该保持不变。

在本网页上注册,即表示您同意,并同意 PacBio 根据我们的隐私政策收集和使用该信息.