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Connecting to databases: exercise part 2

uploading a plasmid to the CARD tool to check for AMR genes
learner's hand working at a computer keyboard with a screen in front

By now you should have an understanding of the main AMR detection tools and what their inputs and outputs look like.

In this exercise, you will download a plasmid sequence from the NCBI sequence repository (separate from their AMR database), upload this sequence to the CARD RGI tool and search for any resistance genes on this plasmid.

Step 1: Get the plasmid sequence

NCBI sequence database entries contain a range of information, including the organism they come from, who uploaded it, what genes may be contained within the sequence.
Have a look through the page and familiarise yourself with its contents.
The main thing to note is the accession number (in this case OM791347). This is needed if you use the public database sequence in your own research, but also can be used to get AMR detection data directly as we see in step 2.
  • We now want to download the fasta format sequence of all the genes on this plasmid.
  • To do this we use the ‘send to’ button at the top right of the page, just under the gray header bar.
Click this and then click ‘coding sequences’ as the type of data you want to download.
Ensure ‘Fasta nucleotide’ is in the dropdown box and then click ‘Create File’
Save this file as plasmid_pD_sequence.txt
If this doesn’t work for you, you can download the file from the bottom of this step page
Step 2: Submit the plasmid sequence to the CARD RGI tool
Input the accession code from NCBI
Upload a fasta format file
  • Click the ‘choose files’ button under the ‘Upload FASTA sequence file(s)’ section.
Select the plasmid_pD_sequence.txt file you just downloaded
At the bottom of the page click ‘submit’
  • Wait a few moments for the task to complete on the web server.
If you get an error, go back to the main RGI page and put the NCBI accession code (OM791347) in the ‘Enter Genbank accession(s)’ box instead and click submit
  • You should now see the results of your search on this plasmid for AMR genes

Proceed to the next step where you can verify your output with some multiple-choice questions.

© Wellcome Connecting Science
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Antimicrobial Databases and Genotype Prediction: Data Sharing and Analysis

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