Species-resolved sequencing of low-biomass or degraded microbiomes using 2bRAD-M

Abstract

Microbiome samples with low microbial biomass or severe DNA degradation remain challenging for amplicon-based or whole-metagenome sequencing approaches. Here, we introduce 2bRAD-M, a highly reduced and cost-effective strategy which only sequences ~ 1% of metagenome and can simultaneously produce species-level bacterial, archaeal, and fungal profiles. 2bRAD-M can accurately generate species-level taxonomic profiles for otherwise hard-to-sequence samples with merely 1 pg of total DNA, high host DNA contamination, or severely fragmented DNA from degraded samples. Tests of 2bRAD-M on various stool, skin, environmental, and clinical FFPE samples suggest a successful reconstruction of comprehensive, high-resolution microbial profiles.

Species-Resolved Metagenomics of Kindergarten Microbiomes Reveal Microbial Admixture Within Sites and Potential Microbial Hazards

“…Carrier RNA was added to increase the yield, and extracted DNA was eluted in the 20 μl RNase-free water. After DNA extraction, 2bRAD libraries were prepared according to Sun et al (2022) . Briefly, 4 U of BcgI (NEB, United States) restriction enzyme was used to digest the genomic DNA at 37°C for 3 h. Next, ligation reaction with 0.2 μM of library-specific adaptors (Ada1, Ada2) at 4°C for 16 h was performed before heat inactivation at 65°C for 20 min for BcgI.…”

“…This yielded high-quality BcgI enzyme-digested sequence fragments of 32 bp, yielding a total of 279 million reads with an average of 2.48 million reads per sample. Taxonomic profiles were determined using the 2bRAD-M computational pipeline; GitHub: https://github.com/shihuang047/2bRAD-M ( Sun et al, 2022 ). Briefly, reads were mapped against a pre-determined 2bRAD tag database which contains taxa-specific BcgI-derived sequences identified from 173,165 RefSeq microbial genomes.…”

“…Unlike human microbiomes, samples collected from the built environment are often of low biomass and can be heavily degraded, thus applying metagenomic sequencing techniques on such samples has been difficult ( Horve et al, 2020 ). To tackle such challenges, we have recently established a new metagenome reduction sequencing approach called 2bRAD-M and showed that it is particularly advantageous for profiling low-biomass, degraded or contaminated microbiomes at the “species” resolution ( Sun et al, 2022 ). Therefore, employing 2bRAD-M and using kindergarten as a model for indoor environment, here we profiled the surface-associated microbiomes at the species resolution and probed possible children–environment–microbe interactions.…”

The renal pelvis urobiome in the unilateral kidney stone patients revealed by 2bRAD-M

“…We therefore sought to apply a method which can produce accurate and species-resolution taxonomic profiles to characterize the low-biomass urobiome in KSD. 2bRAD sequencing for Microbiome (2bRAD-M) is a novel sequencing approach to study the microbiome [ 16 ]. This method digests the genomic DNA of the samples using type IIB restriction enzymes to yield iso-length DNA fragments [ 17 , 18 ].…”

“…The fragments were amplified for sequencing and mapped into species-specific 2bRAD markers for microbial characterization and quantification. 2bRAD-M has proven its ability to profile the low-biomass microbiome at the species level with high fidelity and low costs [ 16 ].…”