New Sequencing Method Provides Full Microbiome “Landscape” at Lower Cost

A research team from the Chinese Academy of Sciences (CAS) has developed a low-cost metagenomic sequencing technique that overcomes the cost and taxonomic resolution challenges of its predecessors and can be used for low biomass, degraded, or contaminated samples.

Metagenomic sequencing is used to identify which microbes are part of a particular microbiome. On method, amplicon sequencing, is cheap, but only produces information about the members of the microbiome at the taxonomic level of genus. It also can’t identify components like bacteria, fungi, or viruses simultaneously. The other metagenomic sequencing method, WMS, can capture whole DNA sequences of everything in the sample—bacteria, archaea, fungi and viruses—and at a higher taxonomic resolution, but is much more expensive and requires high-quality DNA.

"What we need is a low-cost sequencing method that can enable accurate, species-resolution identification of all microbes at the same time, and can handle low biomass samples," says Sun Zheng, a researcher with the Single-Cell Center at CAS’s Qingdao Institute of Bioenergy and Bioprocess Technology (QIBEBT).

The CAS team says new their method is able to determine the full “landscape” of organisms in a microbiome. Dubbed 2bRAD Sequencing for Microbiome, or 2bRAD-M for short, the technique makes use of restriction site-associated DNA sequencing (RADseq) that utilizes Type IIB restriction enzymes—proteins that cleave DNA at specific sites along the molecule—to digest genomic DNA from stool, skin or environment-surface samples into fragments of 25-33 base pairs. 2bRAD-M sequences only these digested fragments—or about just 1% of the metagenome—and can produce profiles across bacteria, archaea, and fungi, down to the level of species.

"By needing only a single picogram (a thousandth of a nanogram) of total DNA, the process can cheaply and accurately generate high-resolution profiles even for hard-to-sequence samples where there has been high host DNA contamination, or the DNA is severely fragmented or otherwise degraded," says Huang Shi, co-first author of the paper.

The CAS team also demonstrated potential clinical applications of 2bRAD-M, which was able to sequence microbiome profiles from formalin fixed paraffin embedded (FFPE) samples of cervical cancer. FFPE samples are typically small, degraded or contaminated by human DNA. Such profiles can potentially be employed for the early diagnosis of cervical cancers.

The findings were published recently in the journal Genome Biology.