Metagenomics Service for Profiling Microbial Communities (16S rDNA & ITS)
Amplicon deep-sequencing using next generation sequencing (NGS) technologies has become a powerful tool to study the diversity of microbial communities. By sequencing parts of the ribosomal DNA (16S rDNA or ITS) derived from environmental samples NGS can generate unprecedented amount of sequence data permitting rapid and profound analysis of microbial communities. However, the processing and evaluation of NGS sequence data is a challenge due to the large amount of data generated.
Metagenomic studies are commonly performed by analyzing amplicons from the 16S rDNA in prokaryotes or the internal transcribed spacer regions (ITS) in fungi (Figure 1). Both loci form a mosaic of highly conserved and hypervariable regions, the latter being used for phylogenetic classifications. It is yet not possible to sequence amplicons spanning the entire 16S rDNA (~1.3 kb) or ITS regions because the read length of current NGS technologies is limited. Which parts of the 16S rDNA or ITS are best amplified for the profiling is still under debate, and varies depending on the study objectives, experimental design and type of sample. In general, if you aim to get a fine-scale taxonomic resolution, your objective should be to cover as many variable regions as possible in your PCR. The challenge of any profiling project is, however, to carefully balance the trade-off between the taxonomic resolution and possible bias from PCR amplification resulting in an under-representation or loss of taxonomic entities . Often only a pilot study will help to find the best primer set for a specific research question.
Microsynth Competences and Services
One of Microsynth’s core competence areas is the profiling of microbial communities based on 16S rDNA analysis. Microsynth is able to offer its customers a full service covering the entire process from experimental design planning, DNA isolation, PCR amplification and sequencing up to a detailed bioinformatics analysis of the generated data (Figure 2).
DNA Isolation: Either the customer provides isolated DNA or outsources this critical step to Microsynth (>13 years of experience in DNA/RNA isolation from various and demanding matrices like plant material, food or stool).
PCR Amplification: The PCR amplification will follow a two-step PCR protocol using a state-of the-art high-fidelity polymerase. This two-step PCR is applied in order to increase reproducibility and to improve the production of high-quality multiplex amplicon libraries. PCR products are purified, quantified with fluorescence spectroscopy using Picogreen and pooled in equimolar amounts.
NGS Sequencing: Sequencing is done using Illumina MiSeq sequencing technology. MiSeq allows high-throughput profiling at low costs with the advantage of long reads (up to 570 bp).
Bioinformatics Analyses: Depending on customer requirements Microsynth offers either a basic bioinformatics analysis package or an advanced bioinformatics analysis package. Both packages are based on the Qiime software . The basic package provides taxonomic profiles and a-diversity measurements (i.e. diversity of organisms in one sample). The advanced package builds on the basic package and adds b-diversity measurements (i.e. diversity of organisms across samples). Guided by experimental parameters (e.g. different sample conditions like temperature, pH, etc.), the comparative analysis of the advanced package allows to test the experimental hypothesis.
Microbial Profiling with Basic Bioinformatics Package
Data Processing: Input for data analysis are demultiplexed, stitched (combining forward and reverse reads), quality filtered and Chimera cleaned fasta reads are used for further analysis.
Data Enrichment: The Qiime software in combination with a reference database is used to define operational taxonomic units (OTUs) and to assign each Out the taxonomic identity at different taxonomic levels (Figure 3). Output may serve as source for down-stream analysis run by the customer.
a-Diversity: Chao1 and Shannon diversity measurements are calculated. Rarefaction analysis is also performed to estimate if sampling has been exhaustive (Figure 4).
Microbial Profiling with Advanced Bioinformatics Package
The Advanced Bioinformatics Analysis includes the results of the basic analysis. In addition, the microbial community structures are compared against the environmental parameters provided by the customer in a phylogenetic context.
The OTU dataset obtained with the basic bioinformatics package serves as input for further analysis.
Data Enrichment: In a first step a phylogenetic tree is calculated for all OTUs in the dataset (UniFrac). The phylogenetic tree serves as basis for the comparative analysis.
b-Diversity: A qualitative overview of intersample diversity is obtained by means of principal coordinate analysis (PCoA, Figure 5A). In addition, significance of pairwise phylogenetic differences among communities is computed and hierarchical UPGMA based clustering is performed (Figure 5B).
Comparative Analysis: A quantitative assessment is performed to test the experimental hypothesis which answers (i) whether the sample categories differ from each other and (ii) whether OTUs are differentially represented based on sample categories (e.g. different sample conditions).
Primer Systems and Their Possible Effects on Profiling Results
 Berry et al. (2011) Barcoded primers used in multiplex amplicon pyrosequencing bias amplification, App Env Microb, 77: 7846-7849.
 Caporaso et al. (2010) Qiime allows analysis of high-throughput community sequencing data, Nature Methods, 7: 335–336.
 Colwell, R.K. & Coddington, J.A. (1994) Estimating terrestrial biodiversity through extrapolation. Philos T Roy Soc B, 345: 101–118.
 Lozupone, C. & Knight, R. (2005) UniFrac: a New Phylogenetic Method for Comparing Microbial Communities, Appl Environ Microbiol, 71: 8228-8235.