Amplicon sequencing is frequently used to identify and differentiate microbial species. Short (<500 bp) hypervariable regions of conserved genes or intergenic regions are amplified by PCR and analyzed using NGS technology, and the resulting sequences are compared against microbial databases.For bacteria and archaea, the 16S rRNA gene is the most common target for amplicon sequencing. For fungi, three targets are generally used: the 18S rRNA gene and two internal transcribed spacers (ITS) located between rRNA genes. These regions are usually sufficiently divergent to separate even highly related species, and can sometimes differentiate subspecies.At Novogene, we have sequenced over 170,000 microbial samples for our customers. Our standard bioinformatics analyses include alpha-diversity analysis, OTU analysis, species annotation, beta-diversity analysis, and multi-variate statistical analysis. Applications range from identifying a single species in pure culture to characterizing the microbiota of animals or plants, to comparing species diversity and population structure in various environmental sources or geographic regions. Our specialists can advise you on the appropriate analyses for your project.
The Novogene Advantage
Highly experienced: We have sequenced over 170,000 samples, resulting in nearly 30 published articles.
Outstanding service: We provide high-quality sequencing, an efficient standard workflow, fast turnaround time, and bioinformatics analyses at a cost-effective price.
Effective methodology: Our method features high amplification efficiency of sample DNA (>95%) .
Comprehensive analysis: We provide expert bioinformatics analyses using the latest sequence databases and software, generating high-quality, publication-ready data.
Project Workflow
Sequencing Strategy
130-470 bp insert DNA library
Ion S5XL, Single-end 400/Single-end 600
Target
Region
Fragment Length
Primer
Primer sequences (5’- 3’)
Bacterial
16S rDNA
V4
292 bp
515F
GTGCCAGCMGCCGCGGTAA
806R
GGACTACHVGGGTWTCTAAT
V3-V4
466 bp
341F
CCTAYGGGRBGCASCAG
806R
GGACTACNNGGGTATCTAAT
V4-V5
393 bp
515F
GTGCCAGCMGCCGCGGTAA
907R
CCGTCAATTCCTTTGAGTTT
Archaeal
16S rDNA
V4
397 bp
519F
CAGCCGCCGCGGTAA
915R
GTGCTCCCCCGCCAATTCCT
288 bp
U519F
CAGYMGCCRCGGKAAHACC
806R
GGACTACNSGGGTMTCTAAT
Fungal
18S rDNA
V4
179 bp
528F
GCGGTAATTCCAGCTCCAA
706R
AATCCRAGAATTTCACCTCT
V9
131 bp
1380F
CCCTGCCHTTTGTACACAC
1510R
CCTTCYGCAGGTTCACCTAC
Fungal
ITS*
ITS1
307 bp
ITS5-1737F
GGAAGTAAAAGTCGTAACAAGG
ITS2-2043R
GCTGCGTTCTTCATCGATGC
ITS2
386 bp
ITS3
GCATCGATGAAGAACGCAGC
ITS4
TCCTCCGCTTATTGATATGC
* ITS1 is located between the 18S and 5.8S rRNA genes; ITS2 is located between the 5.8S and 28S rRNA genes.
Data Quality Guarantee
The amount of data for each sample is not less than 30,000 reads, 50,000 reads or 100,000 reads.
Sample Requirements
Sample Type
Remarks
Amount
Fragment Size
Concentration
Volume
Purity
Genomic DNA
-
≥ 150 ng
-
≥5 ng/μL
≥ 30 μl
OD260/280=1.8-2.0
Turnaround Time
Within 15 working days from verification of sample quality (without data analysis ≤100 samples)
Additional 5 working days for data analysis
Recommended Sequencing Depth
Three strategies: 30,000 reads, 50,000 reads, or 100,000 reads
Analysis Pipeline
List of Analyses
Data quality control
OTUs (Operational Taxonomic Units) clustering and filtering
Alpha-diversity analysis, including rarefaction curve, Chao-1 curve, Shannon curve, rank abundance curve, and alpha indices table
OTUs analysis and species annotation, including Krona results, phylogenetic composition analysis, phylogenetic tree, heatmap, and taxonomic tree
Beta-diversity analysis, including unweighted UniFrac distance heatmap, PCA (principal component analysis), PCoA (principal co-ordinates analysis), UPGMA (unweighted pair-group method with arithmetic means), and NMDS (Non-metric multidimensional scaling) analysis
Multi-variate statistical analysis, including LEfSe (LDA effect size) analysis, metastats analysis, ANOSIM, and MRPP analysis
Project Examples
The following studies utilized Novogene's amplicon sequencing services.The microbiota maintain homeostasis of liver-resident γδT-17 cells in a lipid antigen/CD1d-dependent manner
Nature Communication 8: 13839 (2017)
This study explored how gut microbiota maintain homeostasis of a specific type of T cell in the liver, i.e., liver-resident IL-17A-producing γδT (γδT-17) cells, using Novogene’s amplicon sequencing service to examine microbial diversity. γδT-17 cells are key contributors to maintaining the immune response to microbiota, and comparison of these cells from various organs showed that hepatic γδT cells produce high levels of the pro-inflammatory cytokine IL-17A. When treated with various antibiotics, both the γδT-17 cell number and the bacteria species diversity were maintained homeostasis even though different compositions of microbes were induced. Further study showed that the proliferation of γδT-17 cells is promoted by CD1d presentation of commensal lipid antigens. This study is the first to describe the unique impacts of the gut microbiota on the functions of liver-resident γδT cells.
Figure. 16S rDNA sequencing reveals that commensal microbe load positively correlates with hepatic γδT-17 cell numbers
Continental-scale pollution of estuaries with antibiotic resistance genes
Nature Microbiology, 2:16270 (2017)
Aquatic ecosystems are highly susceptible to anthropogenic impacts, including pollutants such as antibiotic resistance genes (ARGs). The spread of these resistance genes into aquatic environments poses a health risk to human and animal populations. To study the factors shaping the diversity and abundance of ARGs in estuaries, researchers sampled sediment from estuaries in China. High-throughput quantitative PCR determined that over 200 ARGs were present, with 18 found in all sediment samples. Amplicon sequencing of the 16s rRNA gene was performed by Novogene to assess the abundance of bacterial cells and bacterial community composition in sediments, which was found to be consistent across different estuaries, indicating that bacterial composition was not a key contributor to ARG variance. ARG abundance was positively correlated with antibiotic residues and socio-economic parameters, including total population, gross domestic product, and sewage and aquaculture production. These results indicate that human activity is largely responsible for the increased abundance and spread of ARGs in aquatic environments. The study emphasizes the environmental, agricultural, and medical consequences that can arise from such pollution. Additionally, it demonstrates how amplicon sequencing can be used to study microbial communities in aquatic environments.
Figure. ARG profiles in estuarine sediments
Examples of Publications Using Novogene’s Services
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I Understand
is frequently used to identify and differentiate microbial species. Short (<500 bp) hypervariable regions of conserved genes or intergenic regions are amplified by PCR and analyzed using NGS technology, and the resulting sequences are compared against microbial databases.
For bacteria and archaea, the 16S rRNA gene is the most common target for amplicon sequencing. For fungi, three targets are generally used: the 18S rRNA gene and two internal transcribed spacers (ITS) located between rRNA genes. These regions are usually sufficiently divergent to separate even highly related species, and can sometimes differentiate subspecies.
At Novogene, we have sequenced over 170,000 microbial samples for our customers. Our standard bioinformatics analyses include alpha-diversity analysis, OTU analysis, species annotation, beta-diversity analysis, and multi-variate statistical analysis. Applications range from identifying a single species in pure culture to characterizing the microbiota of animals or plants, to comparing species diversity and population structure in various environmental sources or geographic regions. Our specialists can advise you on the appropriate analyses for your project.
