AN_ShotgunMetagenomics

Understand the genetic potential of your community samples Provides you with hypothesis-free taxonomic analysis Introduction Microbiome studies are often based on the sequencing of specific marker genes as for instance the prokaryotic 16S rRNA gene. Such amplicon-based approaches are well established and widely used. However, they have limitations such as the dependency on a single gene to analyze a whole community, the intro - duction of PCR bias and the restriction to describe only the taxonomic compo - sition and diversity. Shotgun metage - nomics is a cutting edge technique for microbiome analysis overcoming said limitations. Whole genomic DNA of a sample is isolated, fragmented and finally sequenced. This allows a detailed analysis of the taxonomic and functional composition of a microbial community. Metagenomics Analysis of Microbiota by Next Generation Shotgun Sequencing Microsynth Competences and Services Microsynth offers a full shotgun metagenomics service for taxonomic and functional profiling of clinical, envi - ronmental or engineered microbiomes. The service covers the entire process from experimental design, DNA isola - tion, tailored sequencing to detailed and customized bioinformatics analy - sis. A major advantage of Microsynth’s shotgun metagenomics service is the project specific consulting and support by our experienced NGS specialists. Experimental Design: Microbiome profiling by shotgun metagenomics often means sequencing of numerous samples generating millions or even billions of reads. To gain reliable and high-quality data, a well-planned experi - mental design including replicates and a clear scientific hypothesis is essential [1]. Our NGS specialists will assist you from the beginning. DNA Isolation: Total DNA is either iso - lated by the customer or this critical step is outsourced. Microsynth has over 15 years of experience in nucleic acid iso - lation from various demanding matrices such as food, stool, eukaryotic tissues (plants, animals, human), water or soil. Tailored NGS Sequencing: Microsynth will tailor the whole sequencing process to your project requirements, thus pro - viding you with just the right amount of data to answer your questions. Bioinformatics: Taxonomic and func - tional analysis of metagenomic data - sets is challenging. Alignment, binning and annotation of the large amounts of sequencing reads require expertise and sufficient computational power. Microsynth offers cutting edge bio - informatics analysis for your shotgun sequencing data using published and well-established methods. Our analysis pipeline will be adjusted to your specific requirements to guarantee scientifically reliable results for your project. After quality processing the reads are aligned against a protein reference database (e.g. NCBI nr). Taxonomic and functional binning and annotation are performed. The analysis is not restricted to prokar - yotes but also includes eukaryotes and viruses. The output comprises all reads, alignments as well as the taxonomic and functional annotation. Together with the raw and processed data, an HTML report is sent, which facilitates the examination, filtering and sorting of the data. Shotgun Metagenomics AnalysisProject Output: Soil Samples DNA Isolation Illumina DNA Library Preparation Illumina 2 x 150 bp Sequencing Option I: Samples Option II: Isolated DNA Project Input: Report Generation Option III: Bioinformatics only Experimental Design Option I: Library Prep only Option II: Raw Data only Option III: Full Report Microsynth AG, SwitzerlandSch?tzenstrasse 15 ? P.O. Box ? CH - 9436 Balgach ? Phone + 41fi71fi722fi83 33 ? Fax + 41fi71fi722fi87 58 ? info @microsynth.ch ? www.microsynth.com THE SWISS DNA COMPANY Application Note ? Next Generation Sequencing Microbiome Data Analysis Examples Microsynth’s analysis pipeline provides you with full flexibility regarding data inspection and analysis. It allows you to analyze and visualize the taxonomic and functional composition of your micro - biome dataset in a single user friendly interface. A major advantage is the pos - sibility to import metadata such as envi - ronmental para-meters or experimental conditions for further statistical analysis. On all levels, data can easily be exported in prevalent formats, giving you the pos - sibility to specifically extract the relevant information from your dataset and use the data for downstream analysis and figure generation. Taxonomic content can be explored on all ranks from domain level down to the species level covering the whole NCBI taxonomy (see Figure 1 ). Functional annotation is based on differ - ent functional classification systems such as InterPro, KEGG, eggNOG or SEED. All systems contain a functional hierarchy ranging from general classifications such as metabolism or cellular functions (see Figure 2 ) down to single enzymes and subunits. Beside the analysis of single samples, multiple sample comparisons are possible (see Figure 3 ). Samples can be grouped and metadata information can be included in the analysis. Various beta diversity measures can be chosen for sample comparison. Further Reading 1. Knight et al. (2012) Unlocking the potential of metagenomics through replicated experimental design, Nat Biotechnol, 30, 513-520. Figure 1. Word cloud representing the bacterial composition on the phylum level in a sample. The size of the words is proportional to the number of assigned reads. Figure 2. Radial tree chart representing the KEGG functions carbohydrate metabolism and energy metabolism and the related subgroups. The size of the sections in each hierarchy is proportional to the number of assigned reads. Figure 3. PCoA for multi-sample comparison based on Bray-Curtis dissimilarities for the InterPro functional annotation. Related Topics • 16S Metagenomics Sequencing • Amplicon Deep Sequencing • Bioinformatics Services • DNA/RNA Isolation Microsynth AG, SwitzerlandSch?tzenstrasse 15 ? P.O. Box ? CH - 9436 Balgach ? Phone + 41fi71fi722fi83 33 ? Fax + 41fi71fi722fi87 58 ? info @microsynth.ch ? www.microsynth.com THE SWISS DNA COMPANY Application Note ? Next Generation Sequencing