THE SWISS DNA COMPANY Application Note · Next Generation Sequencing Metatranscriptome Analysis by Next Generation Shotgun Sequencing Understand the active genetic composition of your samples Functional results for active transcripts and switch on pathways Introduction Shotgun metatranscriptomics is emerg- established reference genomes, compu- files in microbial community across all ing as a powerful technology for the func- tational tools and bioinformatics pipe- species tional characterization of complex micro- lines make analysis and interpretation • Identification of community activities bial communities (microbiomes). While of large sequencing datasets challeng- and functions, e.g. active biochemical metagenomics tells us which microbes ing. Microsynth has become an expert in functions and metabolic pathways are present and what genomic poten- this area and can help you in any aspect • Discovery of novel genes and regula- tial they have, metatranscriptomics tells of a metatranscriptomic sequencing tory pathways us about their activity: the (differential) project. Therefore, approach us in case • Differential gene expression analysis, expression of genes (mRNA transcripts) your application includes one or several e.g. detection of functional changes in in a specific microbial environment at a of the following topics: a given context given point of time. However, the lack of • Determination of gene expression pro- Microsynth Competences and Services Microsynth offers a complete shotgun snapshot of the gene expression profile Library Preparation and Sequencing: metatranscriptomics service for taxo- at the time of sampling, adequate sam- Since a large fraction of total RNA in a nomic and functional profiling of clinical, pling procedures are essential for obtain- cell accounts for ribosomal RNA, deple- environmental or engineered samples. ing an unbiased gene expression profile. tion of ribosomal RNA or poly(A) enrich- The service covers the entire process RNA Isolation: Total RNA is either iso- ment are optional steps after RNA iso- from experimental design, RNA isola- lated by the customer or this critical step lation to increase the fraction of coding tion, tailored sequencing to detailed is outsourced. Microsynth has over 15 RNA. Library preparation includes and customized bioinformatics analy- years of experience in nucleic acid iso- reverse transcription of RNA into cDNA sis. A major advantage of Microsynth’s lation from various demanding matrices and preparation of Illumina shotgun shotgun metatranscriptomics service such as food, stool, eukaryotic tissues, libraries. Microsynth will tailor the whole is the project-specific consulting and water or soil. sequencing process to your project support by our experienced NGS special- Project Input: ists throughout the entire project. Experimental Design Experimental Design: Gene expres- Option I: sion profiling by shotgun metatran- Samples Bioreactor Samples scriptomics often means sequencing of numerous samples generating millions Option II: Isolated RNA Total RNA Isolation or even billions of reads. To gain reliable Project Output: and highquality data, a well-planned Option I: Illumina TruSeq RNA incl. Bacterial Ribo Library Prep only experimental design including replicates Depletion Library Preparation as well as a clear scientific hypothesis is Option II: essential. Our NGS specialists are happy Illumina NextSeq (1 x 150bp) Sequencing Raw Data only to assist you from the very beginning. Option III: Sampling: For each metatranscriptome Bioinformatics Shotgun Metatranscriptomics Analysis only analysis, sampling is the most critical Option III: step. Since the analysis will result in a Report Generation Full Report 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 requirements, thus providing you with using published and well-established also includes eukaryotes and viruses. The just the right amount of data to answer methods. Furthermore, our analysis number of reads mapped to an anno- your questions. pipeline will be adjusted to your specific tated feature (e.g. gene IDs) is the input Bioinformatics Analysis: Taxonomic project requirements in order to yield for the differential gene expression anal- and functional analysis of metatran- scientific reliable results. The reads are ysis. Specialized statistical software pack- scriptomic datasets is challenging. quality processed and aligned against a ages are used to identify differentially Alignment, binning and annotation protein reference database (e.g. NCBI nr) expressed genes. First, data is normalized of large amounts of sequencing reads using DIAMOND, a sensitive tool 20,000 and variance is calculated based on rep- require expertise and sufficient com- times faster than BlastX [1]. Taxonomic licate samples for each condition. Finally, putational power. Microsynth offers and functional binning and annotation differentially expressed genes are identi- cutting-edge bioinformatics analy- are performed by MEGAN [2]. The anal- fied by statistical testing. sis for your shotgun sequencing data ysis is not restricted to prokaryotes but Examples for Metatranscriptome Data Analysis Microsynth’s analysis pipeline provides from your dataset and use the data for examples, please see also our Application you with full flexibility regarding data downstream analysis and figure genera- Note “Metagenomics Analysis of inspection and analysis. The metatran- tion. Differential gene expression analy- Microbiota by Next Generation Shotgun scriptome can be explored and ana- sis results in a log fold change per gene Sequencing” (please click here to be lyzed using the freely available com- for the pairwise comparison between directed to our website section where munity edition of MEGAN. On all levels, two conditions as displayed in Figure 1A. you can download the corresponding data can easily be exported in standard Examples for graphical output are heat- PDF). formats giving you the possibility to spe- maps of the top up- and down-regulated cifically extract the relevant information genes (see Figure 1B). For more analysis Figure 1: Results from the metatranscriptome analysis. 1A. Summary table resulting from differential gene expression analysis of the metatranscriptomic dataset. The IDs refer to the InterPro classification for proteins. The column logFC represents the logarithmic fold change between Condition1 and Condition2. Statistical sig- nificance is given by the p-value and the adjusted p-value for multiple testing. 1B. Heatmap of the top 30 upregulated genes between Condition1 and Condition2. Related Topics Further Reading • Shotgun Metagenomics 1. Buchfink et al. (2015) Fast and sensitive protein alignment using DIAMOND, Nat Methods, 12, 59-60. • RNA Sequencing 2. Huson et al. (2016) MEGAN Community Edition – Interactive Exploration and Analysis of Large-Scale • 16S Metagenomics Sequencing Microbiome Sequencing Data, PLOS Comput Biol, 12, e1004957. • Bioinformatics Services 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.