Mar 28, 2025

Public workspaceCycloneSEQ library construction from DNA of isolated bacteria

DOI
dx.doi.org/10.17504/protocols.io.rm7vzk3k2vx1/v1
  • Wang Mengmeng1,
  • Hewei Liang1,
  • Tao Zeng1,
  • Chen Junyi1,
  • Zou Yuanqiang1,
  • Xiao Liang1
  • 1BGI
Wang Mengmeng
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzk3k2vx1/v1
Protocol Citation: Wang Mengmeng, Hewei Liang, Tao Zeng, Chen Junyi, Zou Yuanqiang, Xiao Liang 2025. CycloneSEQ library construction from DNA of isolated bacteria. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzk3k2vx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 21, 2024
Last Modified: March 28, 2025
Protocol Integer ID: 112526
Keywords: genomics, long-read sequencing, microbiology, CycloneSEQ
Funders Acknowledgements:
The Shenzhen Municipal Government of China
Grant ID: XMHT20220104017, CXB201108250097A
Abstract
CycloneSEQ is a novel long-read sequencing platform developed by BGI-Research This CycloneSEQ library protocol is tailored for the assembly of circular complete genomes from mixed microbial communities. DNA is extracted from bacterial strains and after construction the resulting libraries can be sequenced on the CycloneSEQ platform.
CycloneSEQ library construction from DNA of isolated bacteria
CycloneSEQ library construction from DNA of isolated bacteria
50m
50m
Approximately Amount2 mL of liquid medium with 108 or 109 CFU (colony forming units) of bacterial cells are extracted using a DNA extraction Kit for high-throughput applications.
CycloneSEQ library preparation and sequencing follows the manufacturer’s guidelines. Each sample, containing 2 μg of input DNA (≥21 ng/μL), is diluted with nuclease-free water to Amount192 µL , then mixed with Amount14 µL each of DNA repair buffers 1 and 2, Amount12 µL of DNA repair enzyme 1, and Amount8 µL μL of DNA repair enzyme 2.
The mixtures are then incubated in a thermocycler at Temperature20 °C for Duration00:10:00 , Temperature65 °C for Duration00:10:00 , and held at Temperature4 °C .
20m
After incubation, the mixtures are purified with 1.0x DNA clean beads and eluted with Amount240 µL of nuclease-free water.
The end-repaired samples are then mixed with Amount10 µL of sequencing adaptors, Amount100 µL of 4x ligation buffer, Amount40 µL of DNA ligase, and Amount10 µL of nuclease-free water, and incubated at Temperature25 °C for Duration00:30:00 .
30m
The ligated products are purified again with 1.0x DNA clean beads, resuspended with long fragment wash buffer, and recovered into Amount42 µL of elution buffer.

The resulting libraries are quantified using a Qubit fluorometer and sequenced on the CycloneSEQ WuTong02 platform according to the manufacturers sequencing protocols.