Alternative tools for phylogeny. Identification of unique core sequences

Transcription

1 Alternative tools for phylogeny Identification of unique core sequences Workshop on Whole Genome Sequencing and Analysis, Mar. 2018

2 Learning objective: After this lecture you should be able to account for non-snp based methods for deducing phylogeny account for a method for identification of unique core sequences in a set of strains with a particular phenotype

3 Alternative tools for phylogeny

4 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

5 Database Content (13th of Feb. 2018)......

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7 Morganella tree

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

9 (16s rrna)

10 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. Enterobase (Warwick Medical School): cgmlst and wgmlst for Salmonella, Escherichia/Shigella, and Yersinia CLCBio, BioNumerics, Ridom SeqSphere+ (commercial): offer cgmlst/ wgmlst for a number of species CGE: in the process of implementing cgmlst for Campylobacter, E. coli, Listeria, Salmonella, and Yersinia

11 Campylobacter scheme is from pubmlst Listeria scheme is from Pasteur Institute Salmonella, E. coli, and Yersinia are schemes from Enterobase

12 cgmlstfinder is currently in betamode Only accepts raw reads (fastq files and NOT zipped) For some schemes (e.g., Listeria) it is currently only possible to have alleles identified, no final translation to Sequence Type

13 SNP/ND based methods versus cgmlst/wgmlst 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 free solutions for a few species

14 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

15 Distinguishing E. coli ST-131 outbreak strains from non-outbreak strains 16 isolates from a nosocomial outbreak of ESBL E. coli ST-131 in Sweden was subjected to WGS (Illumina) after PFGE and LMqPCR HRMA were unable to judge if two isolates (no. 8 and 13) were part of outbreak 3 E. coli control strain were also sequenced (no. 17, 18, and 21) The CGE tools MLST, ResFinder, and PlasmidFinder were used to identify the multilocus sequence type and look for antibiotic resistance genes, and plasmid replicons ST aac(60)ib-cr blaoxa blactx-m blatem-1b IncF II Inc FIA Inc FIB Woksepp et al APMIS 125:

16 cgmlst (SeqSphere) was performed on all strains + reference strain JJ1886, which is also ST-131, but not part of the outbreak. cgmlst could confirm that strains 8 and 13 are not part of outbreak

17 And SNP-based phylogeny (CSIPhylogeny) agree

18 Author discussion cgmlst and SNP-based phylogeny were both able to distinguish outbreak from non-outbreak strains cgmlst has the advantage of making communicating typing results between laboratories possible There is still a need to establish cgmlst as a tool in clinical practice and standardization of cgmlst is urgently needed Although resulting in the same clustering in this case, reference-based SNP analysis of highly diverse organism like E. coli might result in false-negative clustering, but may be favourable for, e.g., M. tuberculosis with more clonal evolution In any case SNP-based phylogeny may not be comparable between laboratories or over time (depend heavily on methodology, e.g., reference)

19 Unique core sequences in genomes with specific phenotype Phenotype+ Phenotype+ Phenotype+ Phenotype- Phenotype- Phenotype-

20 RUCS: Rapid Identification of PCR Primers Pairs for Unique Core Sequences Martin Christen Frølund Thomsen, Henrik Hasman, Henrik Westh, Hülya Kaya, Ole Lund; RUCS: rapid identification of PCR primers for unique core sequences, Bioinformatics, 2017, 33(24):

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22 Prepare the data for RUCS: Create a directory/folder called positives and add all positive draft genomes in fasta format (not zipped) Create a directory/folder called negatives" and add all negative draft genomes in fasta format (not zipped) Select both folders and create a zip file (on Mac you right click and select Compress 2 items ) The zipped file is what is uploaded as Isolate File Output example: Unique core sequences (sequences found in all the positive genomes and never in the negative genomes)

23 If you run RUCS in full mode, it will even design PCR primers for the unique sequences

24 Summary For some species cgmlst and wgmlst are alternatives to SNP/ND based methods and offer nomenclature The RUCS method identifies unique core sequences in strains with common phenotype.