QCIF Facility for Advanced Bioinformatics, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
University of Queensland Centre for Clinical Research, Brisbane, Queensland, Australia.
BMC Genomics. 2021 Jun 25;22(1):474. doi: 10.1186/s12864-021-07767-z.
Oxford Nanopore Technology (ONT) long-read sequencing has become a popular platform for microbial researchers due to the accessibility and affordability of its devices. However, easy and automated construction of high-quality bacterial genomes using nanopore reads remains challenging. Here we aimed to create a reproducible end-to-end bacterial genome assembly pipeline using ONT in combination with Illumina sequencing.
We evaluated the performance of several popular tools used during genome reconstruction, including base-calling, filtering, assembly, and polishing. We also assessed overall genome accuracy using ONT both natively and with Illumina. All steps were validated using the high-quality complete reference genome for the Escherichia coli sequence type (ST)131 strain EC958. Software chosen at each stage were incorporated into our final pipeline, MicroPIPE. Further validation of MicroPIPE was carried out using 11 additional ST131 E. coli isolates, which demonstrated that complete circularised chromosomes and plasmids could be achieved without manual intervention. Twelve publicly available Gram-negative and Gram-positive bacterial genomes (with available raw ONT data and matched complete genomes) were also assembled using MicroPIPE. We found that revised basecalling and updated assembly of the majority of these genomes resulted in improved accuracy compared to the current publicly available complete genomes.
MicroPIPE is built in modules using Singularity container images and the bioinformatics workflow manager Nextflow, allowing changes and adjustments to be made in response to future tool development. Overall, MicroPIPE provides an easy-access, end-to-end solution for attaining high-quality bacterial genomes. MicroPIPE is available at https://github.com/BeatsonLab-MicrobialGenomics/micropipe .
由于 Oxford Nanopore Technology（ONT）测序设备的易用性和可负担性，其长读测序已成为微生物研究人员的热门平台。然而，使用纳米孔读取轻松且自动构建高质量的细菌基因组仍然具有挑战性。在这里，我们旨在创建一个使用 ONT 与 Illumina 测序相结合的可重复的细菌全基因组组装管道。
我们评估了在基因组重建过程中使用的几种流行工具的性能，包括碱基调用、过滤、组装和抛光。我们还使用 ONT 评估了细菌基因组的整体准确性，包括使用 ONT 原生数据和使用 Illumina 数据。使用大肠杆菌序列型（ST）131 菌株 EC958 的高质量完整参考基因组对所有步骤进行了验证。在每个阶段选择的软件都被整合到我们的最终管道 MicroPIPE 中。使用 11 个额外的 ST131 大肠杆菌分离株进一步验证了 MicroPIPE，证明可以在无需人工干预的情况下获得完整的环状染色体和质粒。我们还使用 MicroPIPE 组装了 12 个公开的革兰氏阴性和革兰氏阳性细菌基因组（具有可用的原始 ONT 数据和匹配的完整基因组）。我们发现，与当前公开的完整基因组相比，这些基因组的修订碱基调用和更新组装大多数情况下都能提高准确性。
MicroPIPE 使用 Singularity 容器映像和生物信息学工作流管理器 Nextflow 构建在模块中，允许根据未来的工具开发进行更改和调整。总体而言，MicroPIPE 为获得高质量的细菌基因组提供了一个简单易用的端到端解决方案。MicroPIPE 可在 https://github.com/BeatsonLab-MicrobialGenomics/micropipe 获得。
