Scientists propose a high-resolution, high-sensitivity microbiome sequencing technique

Microbiome (also known as flora) sequencing is widely used in ecological health diagnosis, ecological process monitoring, biological resource mining, synthetic biology research and other fields
.

In view of the current pain points and difficulties in the field of bacterial sequencing methodology, the Single Cell Center of the Qingdao Institute of Bioenergy and Processes, Chinese Academy of Sciences and Ocean University of China proposed a high-species resolution, high-sensitivity, and simultaneous identification of all prokaryotic and 2bRAD-M, a low-cost microbiome sequencing technology for eukaryotic microorganisms that is not afraid of sample degradation or contamination
.

In microbiome research, deciphering the species composition of microbial communities mainly relies on two high-throughput methods: amplicon sequencing (16S/18S/ITS) and shotgun metagenomic sequencing (WMS)
.

At present, these two mainstream methods are facing key bottlenecks
.

Amplicon sequencing suffers from amplification bias, off-target amplification, and low species resolution, and is often unable to detect bacteria, archaea, and fungi simultaneously
.

Although shotgun sequencing solves the above problems to a certain extent, it has high requirements on the quality and quantity of sample DNA, so it is usually difficult to analyze trace, highly degraded or seriously contaminated samples, and the cost of sequencing is relatively high
.

　Accordingly, Dr.
Sun Zheng and Huang Shi from the Single Cell Center of Qingdao Energy Institute proposed a "simplified metagenomic sequencing technology" named 2bRAD-M, which effectively overcomes the above-mentioned core defects of amplicon sequencing and shotgun sequencing, and can serve Efficient analysis of trace, highly degraded or heavily contaminated flora samples in humans and the environment
.

Type IIB restriction endonuclease is a type of restriction enzyme that can recognize a specific nucleotide sequence in a double-stranded DNA molecule, and cleaves at a specific distance upstream and downstream of the recognition site to form short fragments (20-33 bp) of equal length.
Endonuclease
.

As a sequencing technology based on the characteristics of type IIB restriction endonucleases, 2bRAD has been applied to the genome research of hundreds of single species including humans, model animals, and marine animals.

.

But a microbiome is often composed of hundreds or thousands of fungal, bacterial and archaeal species, which is far more complicated than analyzing a single species
.

The principle of 2bRAD-M technology is to process the total bacterial DNA samples, amplify and sequence the IIB digested fragments, and then use the theoretical digested sites on various microbial genome sequences as parameters to infer the bacterial community structure
.

In order to verify the technology, the researchers studied the impact of different sources, similarities or complexities, and different experimental methodologies on the qualitative and quantitative analysis of bacterial flora by simulating enzyme digestion data, and solved the problem of high-repetitive short-fragment DNA interference sequence matching.
Question: By using artificial and natural flora samples, the sensitivity, repeatability, resolution, accuracy and preference of 2bRAD-M were verified, and the potential of this method for sequencing various actual flora samples was explored.
and limitations
.

Specifically, studies have demonstrated that the technology is effective in processing low-biomass microbiota samples
.

For example, 2bRAD-M can obtain species-level, high-accuracy, bacterial and archaeal DNA samples with a total DNA of only 1 pg and a length of only 50 bp that is highly fragmented or 99% contaminated with host DNA.
Results of qualitative and quantitative analysis of bacterial and fungal flora structure
.

2bRAD-M performed equally well against real samples such as skin, gut and environment
.

For formalin-fixed paraffin-embedded (FFPE) samples, which are the most common clinical but extremely small amount of bacterial DNA and are highly degraded or contaminated, only a very small amount of FFPE sliced ​​samples (3 cm × 2 μm) are used, 2bRAD-M Microbial species markers that potentially serve the diagnosis of cervical cancer can be captured
.

These findings have methodological implications for basic medical research and clinical practice such as tumor microbiome and immune tissue microbiome
.

Relevant results were published in the journal Genome Biology in the field of genomics
.

The research is supported by the National Natural Science Foundation of China, the National Key R&D Program, the China Postdoctoral Science Foundation, and the Shandong Provincial Talent Project
.

2bRAD-M technology for efficient analysis of trace-degrading microbiomes
.

Type IIB restriction enzymes are like the three-pointed two-edged halberd in the hands of Erlangshen.
They search for the core recognition site on the microbial DNA and simultaneously cut the flanking sequences (corresponding to "three-pointed and two-edged"), thereby generating a large number of equal-length tags.
, through amplification, sequencing and related algorithm calculations, species-level bacterial, archaeal and fungal identification of trace, highly degraded, and severely polluted flora samples can be performed Source: Qingdao Institute of Bioenergy and Processes, Chinese Academy of Sciences