A low-cost metagenome sequencing technology has been developed by scientists from the Chinese Academy of Sciences’ Qingdao Institute of Bioenergy and Bioprocess Technology (QIBEBT). It allows high-resolution disclosure of a microbiome’s whole ‘landscape’ of species, even from low-biomass, damaged, or polluted samples.

The ‘microbiome’ is referred to a community of microorganisms (bacteria, fungi, protists, and viruses) that live in and on people, as well as any other environment. To determine which microorganisms are part of a specific microbiome, researchers must do metagenomic sequencing, the simultaneous sequencing of genetic material from numerous species.

Even state-of-the-art metagenomic sequencing, however, has a lot of critical drawbacks. The procedure is divided into two categories: amplicon sequencing and whole metagenome sequencing (WMS).

Amplicon sequencing is a low-cost procedure. Unfortunately, it only provides information on microbiome members at the genus level of the taxonomy. It is difficult to achieve taxonomic information at the species or strain level. Amplicon sequencing also has the drawback of detecting bacteria, archaea, fungus, and viruses individually rather than identifying them all at once.

WMS can capture whole DNA sequences of all organisms in a sample, including bacteria, archaea, fungi, and viruses, and at a higher taxonomic resolution. This method provides a lot more information, but it’s also a lot more expensive. Moreover, WMS also demands a significant volume of 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, ” revealed SUN Zheng, first author of the paper and a researcher with the Single-Cell Center at QIBEBT.

The researchers discovered the “2bRAD sequencing for Microbiome” technology, often known as 2bRAD-M. This makes use of restriction site-associated DNA sequencing (RADseq), which digests genomic DNA from feces, skin, or environment-surface materials into fragments of 25-33 base pairs (pairs of nucleotides) using Type IIB restriction enzymes (proteins that cleave DNA at specific locations along the molecule). Only these digested fragments—roughly 1% of the metagenome—are sequenced using the 2bRAD-M approach, which can yield profiles spanning bacteria, archaea, and fungus, and not only up to genus but up to species level.

Image Description:Scheme of the 2bRAD-M workflow. In the library preparation module of the 2bRAD-M pipeline, DNA samples were first digested using a type IIB restriction enzyme. The resulting 2bRAD fragments were enriched and amplified for DNA sequencing. In the computational module of the 2bRAD-M pipeline, we employed both prebuilt and sample-specific 2bRAD marker database to perform taxonomic profiling on 2bRAD data. Firstly, all reads were mapped against the default prebuilt unique 2bRAD marker database (2b-Tag-DB) to identify all candidate species in a 2bRAD-M sample. Next, to accurately estimate the abundance of identified species, we increased the number of taxa-specific 2bRAD markers for each candidate species by reconstructing a reduced 2bRAD marker database (sample-specific 2b-Tag-DB) which contains more 2bRAD markers specific to each candidate species than those in the default 2b-Tag-DB. All the 2bRAD sequences were then remapped to this sample-specific 2b-Tag-DB for abundance estimation of candidate species. In principle, the relative abundance of a given species was calculated as the read coverage of all species-specific 2bRAD markers. Image Source: Species-resolved sequencing of low-biomass or degraded microbiomes using 2bRAD-M

“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,” stated HUANG Shi, co-first author of the paper.

The involvement of microbes in cancer formation has been one of the most exciting discoveries in recent years. Using 2bRAD-M, the researchers demonstrated that the microbiome profiles from cervical cancer tissues might be revealed from formalin fixed paraffin embedded (FFPE) samples, which are often poor in quantity, damaged, or contaminated by human DNA. Such profiles might be used to aid in the early detection of cervical malignancies.

Image Description: 2bRAD-M analysis of low-biomass samples collected from underarm, home, and car surfaces. a Heat map shows identified fungal species via 2bRAD-M in the underarm, home, and car samples. b Co-occurrence network of bacterial and fungal species based on 20 underarm samples profiled by 2bRAD-M. Each green circle represents a species, and its size refers to the degree centrality score. Blue/red edges indicate positive/negative Spearman coefficients. Image Source: Species-resolved sequencing of low-biomass or degraded microbiomes using 2bRAD-M

“In the future, we hope to further develop the 2bRAD-M technique and explore its various clinical applications,” told senior author XU Jian, Director of Single-Cell Center at QIBEBT.

“Ultimately, we hope that 2bRAD-M would become a cost-efficient, popular tool for microbiome research and conquer various challenging tasks that are beyond the reach of current techniques,” concluded Prof. WANG Shi from the Ocean University of China, another senior author of the study.

Story Source: Sun, Z., Huang, S., Zhu, P. et al. Species-resolved sequencing of low-biomass or degraded microbiomes using 2bRAD-M. Genome Biol 23, 36 (2022). https://doi.org/10.1186/s13059-021-02576-9

http://english.qibebt.cas.cn/ne/rp/202201/t20220125_299843.html

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