Other tools for typing and phylogeny

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1 Other tools for typing and phylogeny Workshop on Whole Genome Sequencing and Analysis, Mar. 2017

2 Learning objective: After this lecture you should be able to account for tools for typing Salmonella describe the difference between assembly+blastbased prediction methods and mapping based methods account for non-snp based methods for deducing phylogeny

3 Tools for Salmonella

4 SeqSero predicts the serotype of Salmonella isolates Zhang et al J. Clin. Microbiol (5):

5 More on Salmonella Serotyping SeqSero uses the Kaufman and White Scheme Unfortunately, it will often output that it is either one or the other serotype SalmonellaTypeFinder tries to rectify this 1. MLST is determined 2. Serotype is determined according to Kaufman and White 3. If ambiguous serotype, final serotype is determined based on ST Publication under preparation

6 SPIFinder Identification of Salmonella Pathogenicity Islands The engine of the method is similar to ResFinder, VirulenceFinder, SerotypeFinder etc. (assembly+blast) The database is made from the Pathogenicity Island DataBase (PAIDB) SPIFinder is mentioned in Roer L (2016), msystems, 1(3). pii: e

7 PAst predicts the serotype of Whole Genome Sequenced P. aureginosa isolates Thrane SW et al J. Clin. Microbiol. 54(7):

8 Assembly+BLAST-based methods Assembly Raw reads Draft genome Database w. genes of interest The old, trusty method Slow Genes might be missed if at config ends

9 Mapping based methods Mapping (BWA/Bowtie) Raw reads Database w. genes of interest Initially used in SRST2 (Inouye et al., 2014, Genome Med: 6:90) Faster More sensitive -> higher performance Too sensitive -> false positives caused by noise (contamination)

10 KmerResistance - Identification of acquired resistance genes Examines the number of co-occurring kmers between input data and genes in ResFinder database Uses the winner takes it all strategy I) Kmers are only assigned to the gene with the highest kmer matches II) Kmers matching this best hit are removed III) Step I+II are repeated until no more kmers To avoid false positives, a threshold for min. depth and breadth of coverage is introduced The threshold varies according to the depth and breadth of the entire genome as predicted by KmerFinder Clausen et al. (2016). J Antimicrob Chemother. 71(9):2484-8

11 Output from KmerResistance for Ec

co-occurring kmers between input data and database genes

12 MyKmerFinder Upload your own database with genes of interest (like in MyDBFinder) Genes are identified based on co-occurring kmers between input data and database genes Testet with raw reads from Ec and the papg-dsbd database (Ec )

13 Alternative tools for phylogeny NCBI s Pathogen Detection A centralised system integrating data from bacterial pathogens (food, environment, humans) Public health agencies in US and internationally submit sequence data to NCBI NCBI compare the sequences to those already in database and generates phylogenetic tree based on kmers ( Details of the analysis system will be published at a future date ) The aim is to aid traceback investigations and outbreak response Submitted data are released to the public immediately

14 Database Content (9th of Mar 2017)......

15 Morganella tree (9th Mar.)

16 Ribosomal MLST (rmlst) Jolly et al Microbiology. 158(pt 4):

17 (16s rrna)

core genome MLST (cgmlst) and whole genome MLST (wgmlst) Examining thousands of genes => higher discriminatory power Prefixed set of conserved genomewide genes for each

18 core genome MLST (cgmlst) and whole genome MLST (wgmlst) Examining thousands of genes => higher discriminatory power Prefixed set of conserved genomewide genes for each species Potential for standardisation and a name Nisseria meningitides MLST loci versus cgmlst loci. cgmlst/wgmlst is commercially offered by CLCBio, BioNumerics, Ridom SeqSphere+

19 SNP/ND based methods versus cgmlst/wgmslt Pros Cons SNP/ND based phylogeny Available for all species Free web-services are available (e.g., CGE, NCBI s pipeline) Computationally heavy (slow), if many isolates are included No standardisation, e.g., result may differ if a different reference strain is used No nomenclature cgmlst/ wgmlst Offers standardisation and a name Curated databases for each species must first be made and agreed upon Currently only commercial solutions

20 Increasing discriminatory power Analysis Number of genes sampled Example of nomenclature Discriminatory power SNP/ND everything, also - intergenic regions wgmlst >3000 wst543 cgmlst cst832 rmlst 53 rst512 MLST 5-10 ST698 16S rrna 1 E. coli

21 Summary CGE has more tools available than what we cover here, e.g. tools for Salmonella typing Mapping-based methods are faster and more sensitive than assembly+blast based methods, but care needs to be taken to prevent falsepositives For some species cgmlst and wgmlst are alternatives to SNP/ND based methods and offer nomenclature

Alternative tools for phylogeny. Identification of unique core sequences

Alternative tools for phylogeny Identification of unique core sequences Workshop on Whole Genome Sequencing and Analysis, 19-21 Mar. 2018 Learning objective: After this lecture you should be able to account