Exercise2

In this exercise you will either work with data from Harris et al 2013 (if you are in the MRSA1 or MRSA2 group) or Grad et al 2012 (if you are in the Ecoli1 or Ecoli2 group). The purpose is to examine, how much of their findings we can re-find using the CGE web-services. Tomorrow we will continue to work with this data.

Data

Each group will work with draft assemblies from five strains as specified below. All files are available on the USB stick handed out earlier.

MRSA1-group

Day_1_and_2/MRSA/ERR070039-P11.fsa

Day_1_and_2/MRSA/ERR108054-P15.fsa

Day_1_and_2/MRSA/ERR128707-P26.fsa

Day_1_and_2/MRSA/ERR128718-HW.fsa

Day_1_and_2/MRSA/ERR070041-not-outbreak.fsa

MRSA2-group

Day_1_and_2/MRSA/ERR070040-P12.fsa

Day_1_and_2/MRSA/ERR124435-P24.fsa

Day_1_and_2/MRSA/ERR124434-P22.fsa

Day_1_and_2/MRSA/ERR128716-HW.fsa

Day_1_and_2/MRSA/ERR070035-not-outbreak.fsa

Ecoli1-group

Day_1_and_2/Ecoli/escherichia_coli_ec11-4632_c1.fsa

Day_1_and_2/Ecoli/escherichia_coli_ec11-5538.fsa

Day_1_and_2/Ecoli/escherichia_coli_c236-11.fsa

Day_1_and_2/Ecoli/escherichia_coli_ec11-3677.fsa

Day_1_and_2/Ecoli/escherichia_coli_ec09-7901.fsa

Ecoli2-group

Day_1_and_2/Ecoli/escherichia_coli_ec11-4623.fsa

Day_1_and_2/Ecoli/escherichia_coli_ec11-5537.fsa

Day_1_and_2/Ecoli/escherichia_coli_c227-11.fsa

Day_1_and_2/Ecoli/escherichia_coli_ec11-3798.fsa

Day_1_and_2/Ecoli/escherichia_coli_ec04-8351.fsa

Analysis (all groups)

Confirm the species as either S. aureus or E. coli for 1-2 isolates using KmerFinder.

Again, if you don't want to wait for the jobs to finish, go to the page with links to all the results.

Determine the Sequence Type of each of the five isolates you are working with using the MLST tool. Ecoli groups should choose the E.coli #1 scheme. For Ecoli groups, it is enough to determine the ST for 1-2 isolates, as they are all the same ST.

Analyse the five isolates with ResFinder to assess which transferable (acquired) resistance genes the isolates contain.

Use PlasmidFinder to determine which plasmids the isolates contain. If you are working with the E. coli strains, select the database for Enterobacteriaceae, if you are working with the MRSA strains select the database for Enterococcus, Streptococcus, and Staphylococcus.

Note: The paper referenced at the bottom of the PlasmidFinder input page actually only describes the method when using the Enterobacteriaceae database. The database for Gram-positive plasmids contain previously published replicases (https://www.ncbi.nlm.nih.gov/pubmed/19879906 and https://www.ncbi.nlm.nih.gov/pubmed/22685157), mostly from Enterococci, Streptococci, and Staphylococci.

Save the results from all analyses. They should be used for tomorrow's participant presentations and discussions. Note that the links to the result pages will be working for 1 week only. After that the results will be deleted from the CGE servers and you will not be able to retrieve them.

Compare your results to what is reported in the two papers (The MRSA1 and MRSA2 groups compare to Harris et al 2013, while the Ecoli1 and Ecoli2 groups compare to Grad et al 2012). Here are some questions you might want to focus on:

  • Was the species correctly identified?
  • Did the ST you found correspond to the one listed in the paper? In case of imperfect matches to database alleles, describe the type of mismatch and if it is likely to be a true biological difference in the input genome or due to errors introduced during sequencing or assembly.
  • Which antibiotic resistance genes did you identify, and did they correspond to the antibiotic resistance profile of the isolates according to the paper?
    • If you are working with MRSA isolates, see Table S1 in the supplementary material - you might want to fill out a similar table yourselves: PDF or XLSX
    • If you are working with the E. coli isolates, see page 1, top of second column in the supplementary material or recap Grad et al. You might want to fill out a table for better overview: PDF or XLSX
  • Which possible reasons could there be, if one of the isolates that according to your analyses is susceptible to a specific antimicrobial agent turns out to be resistant, when tested phenotypically?
  • Do the identified plasmids correspond to what is reported in the papers? If you are working with the MRSA isolates discuss which plasmid is most likely to carry the ermC gene. If you work with the E. coli isolates, identify the plasmid, which is most likely to carry the extended-spectrum beta-lactamase (blaCTX-M-15).