To address these challenges, here, we proposed the “2bRAD sequencing for Microbiome” (2bRAD-M) method that utilizes Type IIB restriction enzymes to produce exclusively iso-length DNA fragments of 25–33 bp (depending on the selection of enzymes) for sequencing. Therefore, it remains unclear whether RADseq can provide accurate, landscape-like taxonomic profiles for low-biomass and severely degraded microbiome samples. Moreover, RADseq has not been thoroughly and systematically benchmarked against marker gene-based or WMS approaches. Although RADseq has demonstrated the ability to produce species-resolution microbiome profiles with lower costs, the large size variation of DNA fragments after enzyme digestion results in a high bias in PCR-based amplification and thus the low fidelity of reconstructed taxonomic profile. Restriction site-associated DNA sequencing (RADseq) that utilizes restriction enzymes to digest genomic DNA from a broad range of organisms and sequences only the digested fragments has been applied to genotype variable genetic markers as well as for microbial species detection. Therefore, new methods should be developed that cost-efficiently produce accurate, species-resolution, landscape-like taxonomic profiles for challenging samples like low-biomass, high host-contaminated, and degraded microbiomes. Furthermore, WMS is typically much more costly, due to the much higher sequencing volume required for covering the whole genome (instead of just the marker gene). However, although 1 ng is applicable via certain specialized kits, WMS usually requires a high amount of DNA as the starting material (≥50 ng preferred, 20 ng at a minimum) and moreover is not efficient in tackling DNA samples that are low in biomass, heavily degraded, or dominated by host DNA. In contrast, by sequencing the total DNA, WMS can resolve species- or strain-level taxonomy and offers a landscape-like view that includes all domains of organisms. Although less costly, marker gene analyses can be limited in taxonomic resolution (i.e., at the genus level) and susceptible to PCR bias in composition and abundance estimates moreover, they are usually unable to capture a landscape-like view that includes bacteria, archaea, fungi, and virus due to the lack of universal primers. Metagenome sequencing, widely used to derive the taxonomic profile of microbiome, typically adopts two strategies that target (i) amplicons of phylogenetic “marker genes” (e.g., 16S rRNA for bacteria and archaea, and 18S rRNA or internal transcribed spacer (ITS) for fungi) or (ii) the whole genomes (whole metagenome shotgun WMS).

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