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Amplicon

Amplicon


Product Introduction

(1)16S rDNA    
16S rDNA is the corresponding DNA sequence in prokaryotic genomes that encodes ribosomal 16S rRNA molecules, including conserved regions and hypervariable regions. By comparing the hypervariable regions, different species can be distinguished and the degree of difference between them can be calculated. 16S rRNA hypervariable region generally has 9 regions, each region can reflect the difference between species to a certain extent, but the degree of reflection is different. One or two consecutive hypervariable regions can be sequenced and compared to the database for species screening.   
It can also be based on PacBio three generation sequencing platform sequencing, combined with bioinformatics analysis, to study the differences of microbial diversity and community composition. Compared with the second generation sequencing, only 1~2 hypervariable regions are selected for sequencing, and the full-length 16S rDNA sequence can be obtained by the third generation sequencing. More variation region information can improve the resolution of species identification, improve the accuracy of identification of community composition, and restore the microbial community structure in the sample more truly. 

Schematic Diagram of 16S rDNA Structure and Primer


(2)18S rDNA    
18S rDNA is a DNA sequence in eukaryotes that encodes the small ribosomal subunit rRNA, which contains both conserved regions and hypervariable regions (V1-V9, no V6 region). The conserved region reflects the genetic relationship between biological species, while the variable region can reflect the differences between species, which is suitable for the classification criteria of species and above. In the molecular ecological research of eukaryotes (such as plants, animals, fungi, proto-plants, protozoa, etc.), V4 region is the best choice for 18S rRNA gene analysis and annotation, with the most used database information and the best classification effect. 

Schematic diagram of 18S rDNA structure and primers

    
(3)ITS    
Its full name is the internal transcribed spacer, which is generally located between the sequences of the large and small subunit ribosomal RNA genes on the chromosome. In bacteria and archaea, ITS is located between the 16S and 23S rRNA gene sequences. In eukaryotes, there are two ITS fragments, ITS1 and ITS2. ITS1 is located between 18S and 5.8S rRNA genes, while ITS2 is located between 5.8S and 25S (plant) or 28S (animal) rRNA genes. ITS1 in eukaryotes corresponds to ITS in bacteria and archaea. ITS2 originated from the insertion of its ancestral 23S rRNA gene sequence.   
In the molecular ecology of fungi, the ITS region is currently the most used sequencing target region. We recommend use the ITS2 region for amplification, and select ITS3(5 '-GCATCGATGAAGAACGCAGC-3') and ITS4(5 '-TCCTCCGCTTATTGATATGC-3') as amplification primers. 

Schematic diagram of ITS structure and primers

    
(4) Functional genes   
The so-called functional microorganisms are a class of microorganisms that have received widespread attention due to the importance of their functions in nature, such as nitrifying bacteria, denitrifying bacteria, ammonia-oxidizing bacteria, sulfate-reducing bacteria, carbon-fixing bacteria, and nitrogen-fixing bacteria. Each functional microorganism may be very different in taxonomy, but it has similar genes that enable it to perform the same function. Therefore, the genes that enable these functional bacteria to perform this specific function are called functional genes, suchnifH,nirS,NosZ,PmoA,dsrB,ureCandphoDWait. For these functional genes, PCR universal primers were designed for high-throughput sequencing, which can analyze the functional community structure between samples, and combine with the environmental factors of each sampling point, which can analyze the relationship between environmental factors and functional community structure at the same time.   
The relationship between the genetic diversity of bacterial and fungal communities in the environment and their living environment has been gradually revealed in scientific research over the years, but there are still few studies on the genetic diversity of a wide and large number of phages on the earth. Phage has a variety of highly conserved functional gene fragments, so primers can be designed for the conserved region and identified according to the variable region, PCR universal primers can be designed, and high-throughput sequencing can be carried out to explore the diversity of phage, which is of great significance to clarify the composition of bacterial community structure in the regulatory ecosystem. 

Technical parameters

Products

sequencing strategy

Data volume

16S/18S/ITS/functional gene/high-throughput sequencing

NovaSeq PE250

/MiSeq PE300

6w/3w raw reads

Three-generation full-length amplicon sequencing

PacBio sequel IIe

10K CCS raw reads

Three generations full-length advantage

  • Extra-long reading: full-length 16S rRNA was measured, and species classification was accurate to "species";
  • High resolution: the annotation rate of species level is as high as 85%, and the resolution of species identification is greatly improved;
  • High accuracy: CCS sequence self-correction, base accuracy up to 99.9%;
  • No GC preference: The sequencing process has no GC preference and the data results are more reliable.

Experimental process

Sample DNA extraction
PCR Amplification and Product Purification
Library Construction
Library Quality Inspection
on-board sequencing

Analysis process

Diagram of microbial diversity analysis

Analysis results display

 GraPhlAn annotated diagram 

Clustering analysis diagram

 RDA/CCA diagram

LDA score diagram

Case analysis

Case 1: Construction of microbial communities in underground cultural relics driven by multi-domain interactions [1]  
The researchers conducted 16S rRNA and 18S rRNA amplification sequencing, alongside metagenomic sequencing on ancient tomb walls and surrounding soil samples from the Han tomb at Dahuting, which dates back more than 1800 years. The results showed that the microbial community in the Han tomb at Dahuting was composed primarily consisted of bacteria, with Actinomycetes as the dominant group. Multi-domain interactions play an important role in shaping the microbial community of cultural relics. These findings were crucial for understanding the ecological and physiological characteristics of relic microbial communities and supporting long-term protection of relics.  
 

Bacterial and eukaryotic communities dominate the burials

References:Liu W, Zhou X, Jin T, et al. Multikingdom interactions govern the microbiome in subterranean cultural heritage sites[J]. Proceedings of the National Academy of Sciences, 2022, 119(15): e2121141119.  
 

 

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