Study population and ethics statement
For time point 1, stool samples were collected from six adult volunteers living in the Bay Area, CA, USA. This study involving humans was approved by our institutional internal review board (Stanford IRB 42043; principal investigator: A.S.B.), and informed consent was obtained from all participants. For time point 2, the same individuals were recontacted for a second stool donation, 2 years after the initial one.
The samples for time point 1 were included in the publication of Maghini et al.62, and short-read metagenomic sequencing reads are available at the National Center for Biotechnology Information’s Sequence Read Archive under the identifier PRJNA940499.
Sample collection and processing
Stool samples were collected without a preservative and stored at −80 °C. All DNA extractions were performed using the QIAamp PowerFecal Pro DNA Kit (QIAGEN; 51804) according to the manufacturer’s instructions, with the exception of using the EZ-Vac Vacuum Manifold (Zymo Research) instead of centrifugation. DNA concentration was measured using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific) with the dsDNA High Sensitivity kit.
Metagenomic short-read sequencing
The samples from T1 had already been sequenced; therefore, we generated new libraries only for samples from T2. Metagenomic sequencing libraries were pooled, and 2 × 150 bp reads were generated using the NovaSeq 6000 platform (Illumina; 20012850) to a final depth of 6 Gb per sample.
Metagenomic long-read sequencing
All samples from both time points underwent long-read metagenomic sequencing using the ONT platform. DNA fragment distribution was assessed using a TapeStation (Agilent; G2992AA). Samples with apparent fragmentation were cleaned up using a bead-based protocol before library preparation63.
Libraries were prepared using the Native Barcoding Kit V24 (ONT; SQK-NBD114.24) using 1,000 ng of DNA as input. In the pooling step, four samples were combined, resulting in three total libraries. The libraries were loaded onto PromethION R10.4.1 flow cells (ONT; FLO-PRO114M) and sequenced until exhaustion of the flow cells.
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