Advances in Metagenomics and Its Application in Environmental Microorganisms

Abstract

Metagenomics is a new approach to study microorganisms obtained from a specific environment by functional gene screening or sequencing analysis. Metagenomics studies focus on microbial diversity, community constitute, genetic and evolutionary relationships, functional activities, and interactions and relationships with the environment. Sequencing technologies have evolved from shotgun sequencing to high-throughput, next-generation sequencing (NGS), and third-generation sequencing (TGS). NGS and TGS have shown the advantage of rapid detection of pathogenic microorganisms. With the help of new algorithms, we can better perform the taxonomic profiling and gene prediction of microbial species. Functional metagenomics is helpful to screen new bioactive substances and new functional genes from microorganisms and microbial metabolites. In this article, basic steps, classification, and applications of metagenomics are reviewed.

Keywords: antibiotics resistance genes; bioremediation; metagenomics; next-generation sequencing; shotgun sequencing; third generation sequencing.

Figure 1

Sanger sequencing process. (A) Genome DNA is cut into different DNA fragments by ultrasound or enzyme, connected into a plasmid, and transformed into Escherichia coli. (B) The Dideoxy chain termination method is used for sequencing. The reaction system is a mixture of template, primer, DNA polymerase, and dNTPs. Four kinds of ddNTP (ddATP, ddTTP, ddCTP, and ddGTP) with radioisotope marks are, respectively, added to four reaction systems. If ddNTP is incorporated into the oligonucleotide chain, the 2′ and 3′ of ddNTP do not contain hydroxyl groups and cannot form phosphodiester bonds, then the extension of the DNA chain is terminated. (C) Four PCR reaction system products are added to gel electrophoresis, and autoradiography was used to determine the DNA sequence according to the location of electrophoresis.

Figure 2

ROCHE 454 sequencing process. (A) Construction of micro-reaction system. DNA library is constructed by adding an adapter at the ends of DNA fragments. The magnetic beads combined with primers are mixed in the PCR reaction system (DNA fragments, enzymes, dNTPs). (B) Emulsion PCR. PCR microreaction systems are formed by injecting water (PCR mixture) into the oil, and each system contained only one template and one bead. (C) Pyrophosphate sequencing. Pyrophosphate technology is an enzyme cascade chemiluminescence reaction in the same reaction system catalyzed by four enzymes. In each circle of the sequencing reaction, only one dNTP is added. If it just matches the next base of the DNA template, it will be added to the 3′ end of the sequencing primer under the action of DNA polymerase and release a molecular PPi at the same time. Under the action of ATP sulfurylase, luciferase, and apyrase, an enzyme cascade chemiluminescence reaction is triggered by the PPi. (D) Light signal reception.

Figure 3

Illumina sequencing process. (A) DNA library. Breaks the genome DNA to form DNA fragments, adds adapters at both ends, and constructs a single-stranded DNA (ssDNA) library. After adding DNA library into flowcell, DNA fragments will be attached to the surface of flowcell when passing through flowcell. (B) Bridge PCR. Each DNA fragment is clustered in its position. After bridge amplification, each cluster contains many copies of the ssDNA template. (C) SBS: DNA polymerase, primers, and four dNTPs with specific fluorescence are added to the reaction system at the same time. The 3′ end of these dNTPs is connected with an azide group, which can block the incorporation of the next base, so only one base can be extended at a time. After washing off the remaining dNTP and enzyme with water, photos are scanned. After scanning, add chemical reagent, cut off the azide group and quenching fluorescence, and next cycle. (D) Fluorescent signal reception.

Figure 4

ABI sequencing by oligonucleotide ligation and detection (SOLiD). (A) Emulsion PCR. After the emulsion is amplified, a large number of amplified products from the DNA template are attached to the magnetic beads. Then, the magnetic beads are deposited on the glass slide. (B) SLB. The first circle of sequencing reaction includes: the sequencing primer is combined with the template; the probe is combined with the template; the ligase connects the sequencing primer and the probe, excites the probe fluorescence, and removes the last three bases of the probe. Then plus the second probe, connects the two probes with the ligase, excites the probe fluorescence, and removes the last three bases of the probe. Repeat the above reaction to synthesize the whole chain. (C) Solid template shift. After cutting off the whole synthetic ligation, add sequencing primer n-1, repeat the above steps and start the second circle of reaction. Unidirectional solid sequencing requires five circles of sequencing reactions. The primers are n, n-1, n-2, n-3, and n-4, respectively, and the bases at each position are detected two times. (D) Fluorescent signal reception and interpretation of results. (E) Two-base encoding principle. ① “two-base encoding” specifies the corresponding relationship between different base pairs and four probe colors in the coding region (e.g., red fluorescence represents AT, TC, CA, GT). ② Four bases. ③ The probe is an 8-base single-chain fluorescent probe. The base pairs at positions first and second of the probe are determined, positions from third to fifth are random bases, which can be any of the four bases of “A, T, C, and G,” and positions from sixth to eighth mark four fluorescent dyes of different colors.

Figure 5

Ion torrent process. (A) Emulsion PCR. After the emulsion is amplified, a large number of amplified products from the DNA template are attached to the magnetic beads. Magnetic beads are fixed in the micropores of the semiconductor chip, and each micropore can only contain one magnetic bead. (B) SBS. Fix the ssDNA in the micropore of the semiconductor chip and add different dNTPs in turn. When the DNA polymerase polymerizes the nucleotide onto the DNA strand in the extension, it will release an H⁺, causing the change of pH value and voltage in the reaction system. (C) The electrical signal is accepted and converted into base information.

Figure 6

Nanopore sequencing process. Nanopore sequencing mainly uses nanopore protein, also known as reader protein, which is a transmembrane channel protein. Different voltages are applied on both sides of the membrane. DNA double strand is decoiled under the traction of motor protein, and a single molecule is driven by electrophoresis to pass through reader protein one by one. Different electrical signals are produced and detected when the bases of ATCG pass through.