AN_MicrobialReSeq

Search for virulence and resistance genes and check for mutations Validate your bacterial production stem Characterize microbial isolates by MLST Introduction Due to the emergence of powerful next generation sequencing (NGS) technol - ogies, it has never been easier to rese - quence whole genomes. In case a fully annotated reference genome exists, resequencing may be used to compare differences between genomes of indi - viduals from the same species (please see our de novo service if no reference exists). Using results from such measure - ments, researchers may search for viru - lance, resistance and toxin genes, study inherited or acquired mutations and characterize microbial isolates by multi locus sequence typing. Besides offering our customers state-of-the-art NGS plat - forms, Microsynth has also made signif - icant investments in the bioinformatics analysis area. This application note will give you an overview of Microsynth’s various services in the field of micro - bial resequencing as well as its possible impact on your research. Resequencing of Prokaryotic Organisms Microsynth’s Competences and Services With more than 10 years of experience in the field of next generation sequencing, one of Microsynth’s core competences is to provide high quality one-stop ser - vices from experimental design to bio - informatics analysis. You may either out - source the entire analysis or only single steps to us as illustrated in Figure 1 . Experimental Design Microsynth’s NGS specialists will help you define suitable experimental setups for your resequencing projects and discuss possible strategies to address your research questions. DNA Isolation You may either perform the DNA extrac - tion yourself or outsource this critical step to Microsynth. We have long-stand - ing experience in processing various sample matrices and DNA/RNA sources. Library Preparation and Sequencing Following a quality check of your samples, Microsynth will construct Illumina libraries including specific adaptors with barcodes. Depending on the experimental design, the libraries are pooled and sequenced on the Illumina NextSeq 500/550 platform. By offer - ing datapackages for this service, flex - ible sequencing of any sample number is possible. Figure 1. Microsynth’s workflow for resequencing projects. The workflow can be entered and exited at various steps depending on the requirements of the customer. Resequencing AnalysisProject Output: Samples DNA Isolation Library Preparation Illumina 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 + 41ff71ff722ff83 33 ? Fax + 41ff71ff722ff87 58 ? info @microsynth.ch ? www.microsynth.com THE SWISS DNA COMPANY Application Note ? Next Generation Sequencing Example Results Bioinformatics Analysis Sequencing reads are quality filtered and mapped against the reference genome of the organism to be studied. After further refinement based on best practices, pos - sible single nucleotide variations (SNVs) and small insertions and deletions (InDels) are detected and annotated (see Table 1 ). One or multiple variant callers may be used at once, thus searching in a sensitive or specific way depending on the goals of the study. Whole genome sequencing (WGS) data may also be used for Multi Locus Sequence Type (MLST ) microbial isolates using predefined loci and reference databases (see Figure 2 ). If there is no prior knowledge, sequenced reads may be de novo assembled and predicted genes are screened against community accepted database contain - ing resistance, virulence and toxin genes (see Table 2 ). Beside the sequencing raw data and the output of the bioinformatics analysis, a user-friendly summary report is pro - vided. Provided Output Files: Raw data: Fastq Mapping: BAM/BAI files Variant calling, protein consequences: VCF (for each sample separately) and HTML (includes all samples). Table 1. SNV und InDels - Typical output overview file (HTML) resulting from the variant calling step for SNVs and small InDels. For each sample and chromosome/ contig, the SNV and small InDels are reported separately and the effect of SNVs and small InDels for all annotated features of the reference genome are shown. Besides the html format the data are also given in tab separated format (to import into Excel) and as vcf. Figure 2. MLST subtree: As an illustrative example, a small subtree of all phy - logenetic profiles stored on the PubMLST database [3] of the Cronobacter sequence type is depicted. A customer sample would be placed into its appro - priate phylogenetic context to determine its exact sequence type or its next relative. Sam ple _ID R esis ta n ce g e n es V ir u le n ce g e n es T o xin g e n es Sa m ple _1 f o sB 1 - - S a m ple _2 - a stA 1 - Sa m ple _3 - -TR I1 Table 2. Exemplary cut-out of a table with screening results for resistance, viru - lence and toxin genes. Microsynth AG, SwitzerlandSch?tzenstrasse 15 ? P.O. Box ? CH - 9436 Balgach ? Phone + 41ff71ff722ff83 33 ? Fax + 41ff71ff722ff87 58 ? info @microsynth.ch ? www.microsynth.com THE SWISS DNA COMPANY Application Note ? Next Generation Sequencing