Wang, X.; Zhou, W.; Gou, W.; Zheng, J.-S.

While the Human Microbiome Project (HMP) significantly advanced our understanding of the human microbiome, our revisiting of its fecal ITS2 sequencing data reveals two significant issues. First, technical variations in sequencing or data processing introduce batch effects in downstream analyses. Second, we discovered a mismatch between the reported and actual amplification primers, which requires careful consideration when interpreting, reusing, or referencing HMP ITS2 data and methodology.