Nov 13, 2024
CycloneSEQ library construction from single bacteria
This protocol is a draft, published without a DOI.
- Wang Mengmeng1,
- Liang Hewei1,
- Tao Zeng1,
- Chen Junyi1,
- Zou Yuanqiang1,
- Xiao Liang1
- 1BGI Research
Protocol Citation: Wang Mengmeng, Liang Hewei, Tao Zeng, Chen Junyi, Zou Yuanqiang, Xiao Liang 2024. CycloneSEQ library construction from single bacteria. protocols.io https://protocols.io/view/cycloneseq-library-construction-from-single-bacter-drjf54jn
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 12, 2024
Last Modified: November 13, 2024
Protocol Integer ID: 111943
Abstract
In a recent study, we constructed several high-quality complete bacterial genomes using a hybrid assembly method, which combines long-read sequencing from the CycloneSEQ platform and short-read sequencing from the DNQSEQ platform to fill gaps in bacterial draft assemblies.
The workflow of CycloneSEQ library construction for single bacteria can be found in the source paper and the following protocol.
Approximately 2 mL of liquid medium with 108 or 109 cfu of bacterial cells was extracted using the DNA Kit for high-throughput applications.
The CycloneSEQ library preparation and sequencing followed the manufacturer’s guidelines. Each sample, containing 2 μg of input DNA (≥21 ng/μL), was diluted with nuclease-free water to 192 μL, then mixed with 14 μL each of DNA repair buffers 1 and 2, 12 μL of DNA repair enzyme 1, and 8 μL of DNA repair enzyme 2.
The mixtures were incubated in a thermocycler at 20 °C for 00:10:00 , 65 °C for 00:10:00 , and held at 4 °C .
20m
After incubation, the mixtures were purified with 1.0x DNA clean beads and eluted with 240 µL of nuclease-free water.
The end-repaired samples were then mixed with 10 µL of sequencing adaptors, 100 µL of 4x ligation buffer, 40 µL of DNA ligase, and 10 µL of nuclease-free water, and incubated at 25 °C for 00:30:00 minutes.
30m
The ligated products were purified again with 1.0x DNA clean beads, resuspended with long fragment wash buffer, and recovered into 42 µL of elution buffer.
The libraries were quantified using a Qubit fluorometer and sequenced on the CycloneSEQ WuTong02 platform according to the protocol.