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.

人类肠道微生物组的短读宏基因组测序和从头基因组组装可以产生细菌基因组草案，而无需分离和培养。然而，通过短读长测序组装的细菌基因组通常是碎片化的。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，例如移动遗传元件，从而损害了我们对这些元件对重要细菌表型的贡献的理解。尽管长读长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 进行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类粪便样本中提取微克量高分子量 DNA 的方案，该方案适用于下游长读长测序应用。我们还推出了 Lathe (www.github.com/bhattlab/lathe)，这是一种计算工作流程，用于长读长碱基识别、组装、长读长或 Illumina 短读长的共识细化以及基因组环化。总而言之，该协议可以在大约 10 天内从复杂的人类肠道样本中产生高质量的连续或圆形细菌基因组，并需要 2 天的实践台和计算工作。