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Metagenomics Analysis of Microbiota by Next Generation Shotgun Sequencing

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Metagenomics Analysis of Microbiota by Next Generation Shotgun Sequencing THE SWISS DNA COMPANY Application Note · Next Generation Sequencing Metagenomics Analysis of Microbiota by Next Generation Shotgun Sequencing Understand the genetic potential of your community samples Provides you with hypothesis-free taxonomic analysis Introduction Microbiome studies are often based on as the dependency on a single gene to microbiome analysis overcoming said the sequencing of specific marker genes analyze a whole community, the intro- limitations. Whole genomic DNA of as for instance the prokaryotic 16S rRNA duction of PCR bias and the restriction a sample is isolated, fragmented and gene. Such amplicon-based approaches to describe only the taxonomic compo- finally sequenced. This allows a detailed are well established and widely used. sition and diversity. Shotgun metage- analysis of the taxonomic and functional However, they have limitations such nomics is a cutting edge technique for composition of a microbial community. Microsynth Competences and Services Microsynth offers a full shotgun to your project requirements, thus pro- requirements to guarantee scientifically metagenomics service for taxonomic viding you with just the right amount of reliable results for your project. After and functional profiling of clinical, envi- data to answer your questions. quality processing the reads are aligned ronmental or engineered microbiomes. Bioinformatics: Taxonomic and func- against a protein reference database The service covers the entire process tional analysis of metagenomic data- (e.g. NCBI nr). Taxonomic and functional from experimental design, DNA isola- sets is challenging. Alignment, binning binning and annotation are performed. tion, tailored sequencing to detailed and annotation of the large amounts The analysis is not restricted to prokar- and customized bioinformatics analy- of sequencing reads require expertise yotes but also includes eukaryotes and sis. A major advantage of Microsynth’s and sufficient computational power. viruses. The output comprises all reads, shotgun metagenomics service is the Microsynth offers cutting edge bio- alignments as well as the taxonomic and project specific consulting and support informatics analysis for your shotgun functional annotation. Together with the by our experienced NGS specialists. sequencing data using published and raw and processed data, an HTML report Experimental Design: Microbiome well-established methods. Our analysis is sent, which facilitates the examination, profiling by shotgun metagenomics pipeline will be adjusted to your specific filtering and sorting of the data. often means sequencing of numerous Project Input: samples generating millions or even Experimental Design billions of reads. To gain reliable and Option I: high-quality data, a well-planned experi- Samples Soil Samples mental design including replicates and a clear scientific hypothesis is essential [1]. Option II: Isolated DNA DNA Isolation Our NGS specialists will assist you from Project Output: the beginning. Option I: DNA Isolation: Total DNA is either iso- Illumina DNA Library Preparation Library Prep only lated by the customer or this critical step Option II: is outsourced. Microsynth has over 15 Illumina 2 x 150 bp Sequencing Raw Data only years of experience in nucleic acid iso- lation from various demanding matrices Option III: Bioinformatics Shotgun Metagenomics Analysis such as food, stool, eukaryotic tissues only (plants, animals, human), water or soil. Option III: Tailored NGS Sequencing: Microsynth Report Generation Full Report will tailor the whole sequencing process Microsynth AG, Switzerland Schützenstrasse 15 · P.O. Box · CH - 9436 Balgach · Phone + 41 71 722 83 33 · Fax + 41 71 722 87 58 · [email protected] · www.microsynth.com THE SWISS DNA COMPANY Application Note · Next Generation Sequencing Microbiome Data Analysis Examples Microsynth’s analysis pipeline provides the whole NCBI taxonomy (see Figure 1). subunits. Beside the analysis of single you with full flexibility regarding data Functional annotation is based on differ- samples, multiple sample comparisons inspection and analysis. It allows you to ent functional classification systems such are possible (see Figure 3). Samples can analyze and visualize the taxonomic and as InterPro, KEGG, eggNOG or SEED. All be grouped and metadata information functional composition of your micro- systems contain a functional hierarchy can be included in the analysis. Various biome dataset in a single user friendly ranging from general classifications such beta diversity measures can be chosen interface. A major advantage is the pos- as metabolism or cellular functions (see for sample comparison. sibility to import metadata such as envi- Figure 2) down to single enzymes and 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 Figure 1. Word cloud representing the bacterial composition on the phylum level in a sample. The size of the can be explored on all ranks from domain words is proportional to the number of assigned reads. level down to the species level covering Figure 2. Radial tree chart representing the KEGG functions carbohydrate Figure 3. PCoA for multi-sample comparison based on Bray-Curtis dissimilarities for metabolism and energy metabolism and the related subgroups. The size the InterPro functional annotation. of the sections in each hierarchy is proportional to the number of assigned reads. Related Topics Further Reading • 16S Metagenomics Sequencing 1. Knight et al. (2012) Unlocking the potential of metagenomics through replicated experimental design, • Amplicon Deep Sequencing Nat Biotechnol, 30, 513-520. • Bioinformatics Services • DNA/RNA Isolation Microsynth AG, Switzerland Schützenstrasse 15 · P.O. Box · CH - 9436 Balgach · Phone + 41 71 722 83 33 · Fax + 41 71 722 87 58 · [email protected] · www.microsynth.com.
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