Microbiome Analysis by 2bRAD-M

Microorganisms play an important role in maintaining ecosystem balance and diversity, and are closely related to human/animal/plant health, environment and food safety. Marker genes (e.g., 16S rRNA for bacteria and archaea, and 18S rRNA or internal transcribed spacer (ITS) for fungi) or the whole metagenome sequencing (WMS) are widely used to derive the taxonomic profile of microbiome. However, marker gene analyses usually are limited at the genus level and susceptible to PCR bias in composition and abundance estimates. Moreover, they are usually unable to capture information of bacteria, archaea, fungi, and virus simultaneously. Although WMS can resolve species- or strain-level taxonomy, and offers a landscape-like view including all domains of organisms, WMS requires a rather high DNA amount (≥ 50 ng preferred, 20 ng at a minimum) and costs high. Creative BioMart Microbe has developed a “reduction” metagenome-sequencing strategy-2bRAD-M (type IIB restriction enzyme site-associated DNA metagenome sequencing), which simultaneously produces species-level bacterial, archaeal, and fungal profiles for low-biomass, high host DNA contamination, and severely degraded samples.

The principle of 2bRAD-M depends on the digestion of Type IIB restriction enzymes, which produce exclusively iso-length DNA fragments of 21-33 bp (depending on the selection of enzymes) for adaptor ligation, amplification and sequencing. Then the tag sequences can be mapped to reference genomes to reconstruct faithfully the taxonomic composition (Figure 1). For example, BcgI, a commercially available Type IIB restriction enzymes, recognizes the sequence of CGA-N6-TGC in the genomic DNA extracted from microbiome samples and cleaves on both upstream (12-10 bp) and downstream (10-12 bp) of this site, producing an average of 3,010 iso-length (32 bp) 2bRAD tags per genome, and a total of 114,132,487 BcgI-digested unique species-specific 2bRAD tags that are of single-copy within a genome (average 1,949 per species genome).

Figure 1 The Principle and Workflow of 2bRAD-M. (Sun et al., 2021)

The Advantage of 2bRAD-M:

• 2bRAD-M accurately reconstructs a comprehensive species-resolution profile of bacteria, archaea and fungi simultaneously.
• 2bRAD-M is suitable for low DNA amount (merely 1 pg total DNA), high host DNA contamination (99%), and severely degraded DNA (50-bp) (such as formalin-fixed paraffin-embedded (FFPE) tissue samples) samples.
• 2bRAD-M is cost-effective, providing the species-level classification information as metagenomics, but with low sequencing cost.

Workflow of Microbiome Analysis by 2bRAD-M Service in Creative BioMart Microbe

Creative BioMart Microbe's expert team is always able to provide customers with efficient, cost effective, and accurate microbiome analysis by 2bRAD-M services, and has always been a trusted partner of customers. We are devoted to developing customized solution for your sample. If you are interested in our microbiome analysis by 2bRAD-M service, please feel free to contact us. Creative BioMart Microbe looks forward to cooperating with you.

Reference

1. Sun et al., 2020. Species-resolved sequencing of low-biomass microbiomes by 2bRAD-M. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.01.405647