homo sapiens oral microbiome: Ctrl
2bRAD-M microbial metagenomic sequencing was performed by OE Biotech Co., Ltd. (Qingdao, China). Briefly, genomic DNA was digested with Type IIB restriction enzyme BcgI (New England BioLabs, cat. no. R0545) at 37C for 3 hours. The digested fragments were ligated with specific adaptors, then amplified by PCR using Phusion high-fidelity DNA polymerase with dual unique dual index (UDI) barcodes. PCR products were purified by QIAquick PCR Purification Kit (Qiagen, cat. no. 28106) and quantified by Qubit dsDNA HS Assay Kit (Invitrogen, cat. no. Q3258). Equal amounts of barcoded libraries were pooled and sequenced on the Illumina NovaSeq 6000 platform with paired-end 150 bp (PE150) reads. The 2bRAD-M method enables species-resolved taxonomic profiling of low-biomass or degraded microbiomes as described previously (Sun et al., Genome Biology, 2022).