Department of Genetics, Stanford University, Stanford, CA, USA.
Divisions of Hematology and Blood & Marrow Transplantation, Department of Medicine, Stanford University, Stanford, CA, USA.
Nat Protoc. 2021 Jan;16(1):458-471. doi: 10.1038/s41596-020-00424-x. Epub 2020 Dec 4.
Short-read metagenomic sequencing and de novo genome assembly of the human gut microbiome can yield draft bacterial genomes without isolation and culture. However, bacterial genomes assembled from short-read sequencing are often fragmented. Furthermore, these metagenome-assembled genomes often exclude repeated genomic elements, such as mobile genetic elements, compromising our understanding of the contribution of these elements to important bacterial phenotypes. Although long-read sequencing has been applied successfully to the assembly of contiguous bacterial isolate genomes, extraction of DNA of sufficient molecular weight, purity and quantity for metagenomic sequencing from stool samples can be challenging. Here, we present a protocol for the extraction of microgram quantities of high-molecular-weight DNA from human stool samples that are suitable for downstream long-read sequencing applications. We also present Lathe ( www.github.com/bhattlab/lathe ), a computational workflow for long-read basecalling, assembly, consensus refinement with long reads or Illumina short reads and genome circularization. Altogether, this protocol can yield high-quality contiguous or circular bacterial genomes from a complex human gut sample in approximately 10 d, with 2 d of hands-on bench and computational effort.
短读宏基因组测序和从头基因组组装可以在不进行分离和培养的情况下生成细菌基因组草图。然而，从短读测序组装的细菌基因组往往是碎片化的。此外，这些宏基因组组装的基因组通常排除重复的基因组元件，如移动遗传元件，这影响了我们对这些元件对重要细菌表型的贡献的理解。虽然长读测序已成功应用于连续细菌分离株基因组的组装，但从粪便样本中提取足够分子量、纯度和数量的 DNA 用于宏基因组测序可能具有挑战性。在这里，我们提出了一种从人粪便样本中提取微克数量高分子量 DNA 的方案，该方案适用于下游长读测序应用。我们还介绍了 Lathe（www.github.com/bhattlab/lathe），这是一个用于长读碱基调用、组装、用长读或 Illumina 短读进行共识优化以及基因组环化的计算工作流程。总之，该方案可以在大约 10 天内从复杂的人类肠道样本中获得高质量的连续或环状细菌基因组，实际操作和计算时间为 2 天。
