Jun 10, 2025

Public workspaceCycloneSEQ long-read WGS library preparation and sequencing

DOI
https://dx.doi.org/10.17504/protocols.io.n92ldnk2nv5b/v1
CycloneSEQ long-read WGS library preparation and sequencing
  • Qunfei Guo1,
  • youliang Pan1,
  • Wei Dai1,
  • Wei Jiang1
  • 1BGI
Guo qunfei
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DOI: https://dx.doi.org/10.17504/protocols.io.n92ldnk2nv5b/v1
Protocol Citation: Qunfei Guo, youliang Pan, Wei Dai, Wei Jiang 2025. CycloneSEQ long-read WGS library preparation and sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldnk2nv5b/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: June 07, 2025
Last Modified: June 10, 2025
Protocol Integer ID: 219759
Keywords: CycloneSeq, Long-read sequencing, Genomics, WGS, sequencing cycloneseq, libraries on the cycloneseq platform, cycloneseq platform, cycloneseq, sequencing platform, genome, sequencing, read wgs library preparation, extracting dna, dna from animal tissue, wg, dna, read
Funders Acknowledgements:
Nation Key R&D Program of China
Grant ID: 2024TFC3406300
Abstract
CycloneSEQ is a novel long-read sequencing platform developed by BGI-Research. After extracting DNA from animal tissue, we use this protocol to prepare and sequence long-read whole-genome (WGS) libraries on the CycloneSEQ platform.
Troubleshooting
DNA Repair
Sample Transfer and Dilution
  • Transfer 2 μg of DNA sample (concentration ≥23 ng/μL) to a sterile tube.
  • Add nuclease-free water to adjust the total volume to 44 μL.

Repair Mix Preparation
  • Add the following reagents to the diluted DNA:
（1）6 μL DNA repair buffer 1
（2）3 μL DNA repair buffer 2
（3）3 μL DNA repair enzyme 1
（4）4 μL DNA repair enzyme 2
  • Mix thoroughly by gentle pipetting.
Incubation
  • Place in a pre-programmed thermocycler and run:
（1） 20°C for 10 minutes
（2）65°C for 10 minutes
（3）Hold at 4°C (until purification).
Purification of Repaired DNA
Bead Cleanup:
  • Add 1.0x volume DNA clean beads to the reaction mix.
  • Incubate for 5 minutes at room temperature.
  • Pellet beads on a magnetic stand. Discard supernatant.
Elution
  • Wash beads twice with 80% ethanol. Air-dry completely.
  • Resuspend beads in 60 μL nuclease-free water.
  • Transfer supernatant (repaired DNA) to a new tube.
Adaptor Ligation
Ligation Mix Preparation:

Combine:
  • Purified DNA (60 μL)
  • 10 μL Sequencing adaptors
  • 25 μL 4x ligation buffer
  • 10 μL DNA ligase
  • 2.5 μL nuclease-free water
Mix gently by pipetting.

Incubation
  • Incubate at 25°C for 30 minutes.
Purification of Ligated Products
Bead cleanup:
  • Add 1.0x volume DNA clean beads.
  • Incubate 5 minutes at RT. Pellet on magnetic stand. Discard supernatant.
Wash and Elution:
  • Resuspend beads in 500 μL long fragment wash buffer (gentle pipetting).
  • Pellet beads, discard supernatant. Air-dry briefly.
  • Elute DNA in 17 μL elution buffer. Transfer to a clean tube.
Library Quantification and Sequencing
Quantification:
  • Measure library concentration using a Qubit fluorometer.
Sequencing:
  • Load Prepared library onto the CycloneSEQ WuTong02 platform.
  • Run sequencing according to manufacturer specifications.