AN_GenomicEpidemiology

Determine the sequence type of microbial isolates Screen for resistance, virulence, and mycotoxin genes Identify genetic variations that lead to pathogenicity Introduction Genomic epidemiology studies are useful to support public health, combat infectious diseases, maintain food safety, or establish sustainable produc - tion lines. To support our customers in these diverse applications, Microsynth has developed a genomic epidemiology pipeline that allows addressing numer - ous scientific topics concerning your epidemiology project: • Resolve the taxonomy of individual species or sequence type (ST ) • Identify resistance, virulence and myco - toxin genes • Detect multidrug resistant bacterial pathogens (MDR) • Detect acquired mutations and assess their potential functional effects • Establish a population-level profiling and comparison of antimicrobial resis - tance Our genomic epidemiology analysis pipeline is based on next generation whole genome sequencing. The gener - ated data and knowledge will support production lines, can be used to track transmission, or used in pathogen out - break surveillance, to just name a few examples. At Microsynth we will guide you through the whole analysis to assure you will achieve your goals. Genomic Epidemiology Analysis 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 genomic epidemiology analysis 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. Figure 1. Microsynth’s workflow for genomic epidemiology analysis projects. The workflow can be entered and exited at various steps depending on the requirements of the customer. Genomic Epidemiology AnalysisProject Output: Staphylococcus aureus DNA Isolation Illumina TruSeq Library Preparation Illumina NextSeq (2x150bp) 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 Example Results Bioinformatics Analysis Thanks to the use of modern methods and algorithms, hundreds of samples can be analyzed in detail for any genomic epidemiology project. Depending on the focus of the project, many different approaches to bioinformatic analysis are possible, but two example scenarios are described in the next section. In the first example scenario, the genomes contained in the samples come from well-studied model organisms. However, since the composition of the samples is unknown, a shotgun meta - genome analysis is performed first to determine whether the samples contain pure strains or whether meta-communi - ties exist, and in both cases the taxonomy is determined (see Table 1 ). Assuming that the samples contain pure strains of a well-studied micro-organism, one of the established Multi Locus Sequence Typing (MLST ) schemes can be used to determine its sequence type, which goes beyond the determination of the species alone (see Table 2 ). Next, in order to reconstruct the strains contained in the samples, the filtered sequencing reads are de novo assem - bled into contigs on which genes are predicted and annotated. The predicted genes are then screened for homologous sequences among the known resistance, virulence, and mycotoxin genes. Single nucleotide variations (SNVs) and small insertions and deletions (InDels) are determined in comparison to the public reference sequences (see Table 3 ). In the second scenario, the microor - ganisms to be analyzed are neither well studied nor publicly documented. In this case, determining the similarity of the unknown genomes to any of the genomes stored in the RefSeq [1] data - base first and second annotate pre - dicted genes with homologues found for instance in the Pfam [2] database (see Table 4 ) are sensible first steps. If no exact phylogenetic relationship is known for the hundreds of samples involved in a genomic epidemiology project, clus - tering can be used to establish groups of samples that may then be further ana - lyzed on their own [3] (see Figure 2 ). Table 1. This cutout of a result of a shotgun metagenomics taxonomy assignment, shows the composition of the bacterial community found in the analyzed sample. In this case, the sample contains 96 % of the Staphylococcus genus (Tax Level: G) and 92% are identified as Staphylococcus aureus species (Tax Level: S). Table 2. The result of an MLST showing the sequence type of the species found in the sample. In this case, the scheme used for typing included seven genes (arcC, aroE, glpF, gmk, pta, tpi and yqiL) to identify the respective ST and Clonal Complex (CC). The numbers in the gene columns represent different variants of these genes found in the PubMLST database [4]. Table 3. Summary table of the number of observed SNVs and small InDels in the analyzed sample including the type of mutation (silent and non-silent). Percent Tax Level Tax ID Tax Name 100.00 D 2 Bacteria 100.00 P 1239 Firmicutes 100.00 C 91061 Bacilli 96.00 O 1385 Bacillales 96.00 F 90964 Staphylococcaceae 96.00 G 1279 Staphylococcus 92.00 S 1280 Staphylococcus aureus Sample arcC aroE glpF gmk pta tpi yqiL ST Clonal Complex sample1 1 4 1 8 4 4 3 72 CC8 Sample SNV InDels Silent Non-silent sample1 236 12 171 53 Library Preparation and Sequencing Following a quality check of your samples, Microsynth will build Illumina libraries including specific adaptors with barcodes. Depending on the experimen - tal design, the libraries are pooled and sequenced either on the Illumina MiSeq or NextSeq 500/550. These flexible plat - forms allow for optimal sequencing depending on the number of samples and on the required read length. 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 Related Services Microsynth also provides microbial resequencing services that focus on the detection of genetic variations in relation to a refer - ence sequence. References [1] O’Leary NA, Wright MW, Brister JR, Ciufo S, Haddad D, McVeigh R, Rajput B, Robbertse B, Smith-White B, Ako-Adjei D, Astashyn A, Badretdin A, Bao Y, Blinkova O, Brover V, Chetvernin V, Choi J, Cox E, Ermolaeva O, Farrell CM, Goldfarb T, Gupta T, Haft D, Hatcher E, Hlavina W, Joardar VS, Kodali VK, Li W, Maglott D, Masterson P, McGarvey KM, Murphy MR, O’Neill K, Pujar S, Rangwala SH, Rausch D, Riddick LD, Schoch C, Shkeda A, Storz SS, Sun H, Thibaud-Nissen F, Tolstoy I, Tully RE, Vatsan AR, Wallin C, Webb D, Wu W, Landrum MJ, Kimchi A, Tatusova T, DiCuccio M, Kitts P, Murphy TD, Pruitt KD. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res . 2016 Jan 4;44(D1):D733-45 PubMed [2] S. El-Gebali, J. Mistry, A. Bateman, S.R. Eddy, A. Luciani, S.C. Potter, M. Qureshi, L.J. Richardson, G.A. Salazar, A. Smart, E.L.L. Sonnhammer, L. Hirsh, L. Paladin, D. Piovesan, S.C.E. Tosatto, R.D. Finn. The Pfam protein families database in 2019. Nucleic Acids Research (2019) doi: 10.1093/nar/gky995 [3] Lees JA, Harris SR, Tonkin-Hill G, Gladstone RA, Lo SW, Weiser JN, Corander J, Bentley SD, Croucher NJ. Fast and flexible bacterial genomic epidemiology with PopPUNK. Genome Research 29:1-13 (2019). doi: 10.1101/gr.241455.118 [4] Website ( https://pubmlst.org/mlst/ ) sited at the University of Oxford (Jolley et al. Wellcome Open Res 2018, 3:124 [version 1; ref - erees: 2 approved]). The development of this site has been funded by the Wellcome Trust. Figure 2. Clustering of a large numbers of samples with unknown phylogenetic relationship resulting in four distinct groups. Table 4. Detail of a table showing the homologous protein domains and their significance found for the predicted genes of the analyzed sample. Target Name Accession Query Name E-value Score Description of Target TPK_catalytic PF04263.16 gene_8|401_aa|-|7047|8252 0.00038 20.5 Thiamin pyrophosphokinase, catalytic domain CRISPR_Cas9_WED PF18061.1 gene_40|840_aa|-|40414|42936 0.12 12.4 CRISPR-Cas9 WED domain 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