Fig. 1: Workflow of the experimental part of the study.

The figure provides a detailed representation of the workflows conducted in this study. The figure illustrates the workflows conducted in this study, which included: 1) evaluating the efficacy of various DNA isolation kits in terms of quality, quantity, and microbial representation from canine stool samples; 2) comparing library preparation techniques on SRS and LRS platforms for reproducibility; 3) introducing “minitax”, a tool designed to ensure consistent analysis across multiple sequencing platforms; 4) assessing the influence of different databases and tools on microbial profiling; and 5) comparing 16S V1-V9 sequencing on ONT and PacBio platforms to address literature gaps and emphasize bioinformatics workflows. Our goal was to identify reliable procedures for robust and reproducible gut microbiome profiling across both wet-lab and dry-lab methodologies. We performed additional experiments to validate the most extreme experimental and bioinformatics results, particularly focusing on methods that yielded the most inconsistent outcomes in comparison to other techniques. For this purpose, we utilized samples from six additional dogs of various genders and ages. The workflow involved: 1) DNA isolation using the Q kit, 2) Library preparation with the PerkinElmer V1-V3 kit, and 3) Analysis of V1-V9 libraries using the EPI2ME software. Furthermore, we carried out experiments employing a Microbial Community Standard (MCS; Zymo Research D6300) as well as a Gut Microbiome Standard (GMS; Zymo Research D6331) to validate the effectiveness of the four DNA isolation kits used. This included: 1) DNA isolation using the kits applied for the dog samples, 2) Preparation of V1-V2 and V1-V9 libraries, and 3) Sequencing on the corresponding Illumina and ONT platforms based on the library. Created in BioRender. BioRender.com/k32q619.