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16S/18S/ITS Amplicon Sequencing

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CD Genomics is the expert in the field of 16s/18s rRNA and ITS amplicon sequencing service. We offer cheaper, faster and advanced amplicon sequencing technology with Illumina high-throughput sequencing instruments to help you investigating the microbial community diversity. By having been cooperating with lots of customers, we can totally meet your project requirements and budgets in microbial taxonomy and molecular phylogeny.

16S/18S/ITS Amplicon Sequencing

An amplicon is a piece of DNA that is the product of PCR amplification of short hypervariable regions of conserved genes or intergenic regions. Because conventional analysis techniques miss the information of non-cultivable microorganisms, analysis of the amplicons using next generation sequencing (NGS) technologies has emerged as a relatively inexpensive and fast tool for studying the diversity of microbial communities in various environmental conditions. We are providing amplicon sequencing service for three types of microbial target genes.

  • 16S rRNA Sequencing

16s rRNA gene is a highly conserved component and the most widely used marker gene to characterize the diversity of archaea and bacteria. The presence of hypervariable regions in the 16S rRNA gene provides a species-specific signature sequence which is very useful to identify the bacteria. Sequencing of 1-2 16S rRNA hypervariable regions (V3-V4-V5) is a common and well-established method for investigating phylogeny and taxonomy of a given sample, especially that from complex microbiomes or environments.

  • 18S rRNA and ITS Sequencing

Fungi comprise a major portion of the biodiversity and biomass in ecosystems and play an essential role in ecological and economic terms as decomposers, symbionts, parasites or pathogens of other organisms and drug or food provider. 18S rRNA gene is the basic component of fungi cells, which has conservative regions and hypervariable regions structurally. Internal transcribed spacer (ITS) is divided into ITS1 and ITS2 and locate between rRNA genes, which has a higher degree of variation than other genic regions of rDNA. They are generally used for obtaining a high taxonomic resolution for fungi, while the ITS region is the most wildly sequenced DNA region for analyzing fungal diversity in environmental samples.

Service Workflow

The typical procedure for our amplicon sequencing service is summarized in Figure 1. Briefly, the illumine sequencing libraries are generated by using PCR, all libraries are purified and size selected to remove unwanted background products. Samples can be tagged with up to 384 barcodes for sequencing. Sequencing is performed using the Illumina MiSeq PE300 or HiSeq PE250. The raw data is cleaned by removal of adapters and low-quality reads.

Procedure for 16S/18S/ITS  Amplicon Sequencing Figure 1. Procedure for 16S/18S/ITS Amplicon Sequencing.

Recommend Sequencing Platform and Sequencing Depth

  • MiSeq PE300 or HiSeq PE250
  • 20 M clean data or 30,000 raw reads per sample

Data Analysis

The sequenced data may be processed using our analysis pipeline (the steps are illustrated in Figure 2) and customized bioinformatics services are also provided.

Overview of the processing and analysis  steps of 16S rRNA gene sequencing data. Adapted from Bromberg <em>et al</em>, 2015 Figure 2. Overview of the processing and analysis steps of 16S rRNA gene sequencing data. Adapted from Bromberg et al, 2015.

Our standard analysis package includes OTUs clustering, Alpha-diversity analysis (rarefaction curve, Chao-1 curve, Shannon curve, rank abundance curve, and alpha indices table), OTUs analysis and species annotation (taxonomic classifications and assignments, phylogenetic tree, Venn graph, Heatmap, and taxonomic tree), Beta-diversity analysis (PCA analysis, PCoA analysis, unweighted UniFrac distance heatmap, UPGMA and NMDS analysis) and so on.

Our advanced analysis package includes LEfSe (LDA effect size) analysis, Wilcoxon test, metastats analysis, ANOSIM, MRPP analysis and function prediction et al.

Sample Requirements

Small number and large number of microbiome samples from a wide variety of sources including human or animals, natural environments and industrial environments can be sequenced and analyzed. Customers may submit environmental samples for bacteria/fungi gDNA extraction services or submit extracted genomic DNA or PCR products. All DNA samples are validated for purity and quantity and subject to quality control prior to processing. The recommended DNA amount for submission is ≥ 1 μg with a concentration of ≥ 10 ng/µl.

16S/18S/ITS Amplicon Sequencing

CD Genomics can offer a full package of 16s rRNA/ITS sequencing service, usually from DNA extraction, PCR amplification, library preparation to sequencing and data analysis, which enables us to reveal the previously hidden diversity of microscopic life. Our 16S rRNA/18S rRNA/ITS metagenomics sequencing offers unprecedented amounts of sequence data permitting rapid and profound diversity analysis of a variety of microbial communities with suitable and cost-effective solutions. You can choose a variety of options regarding the length of amplicons, the hypervariable region, the number of amplicons pooled together, the number of reads of a given amplicon pool, the depth of coverage, and so on. We are also able to provide full-length 16S/ITS rRNA sequencing with the help of PacBio’s SMRT technology. At CD Genomics, with multiple specialists and years of experience in this area, we guarantee you with high-quality data and satisfying results. If you have additional requirements or questions, do not hesitate to contact us, our specialists would like to solve your problems.

Reference

  1. Bromberg, J. S. et al. Microbiota—implications for immunity and transplantation. Nat. Rev. Nephrol. 2015, 11(6):342-53
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