TUTORIAL: Generating diagnostic primers using the Uniqprimer Galaxy Workflow

Transcription

1 TUTORIAL: Generating diagnostic primers using the Uniqprimer Galaxy Workflow In this tutorial, we will generate primers expected to amplify a product from Dickeya dadantii but not from other strains of Dickeya. Part I. Selection and batch download of genome assemblies from Genbank. If you already have FASTA or multifasta files of your diagnostic target genomes and nontarget genomes, proceed to Part II. In Uniqprimer, include genomes should result in a positive test from the diagnostic primers, and exclude genomes should result in a negative test. We recommend selecting include genomes of three or more bacterial strains that represent the diversity of the target species or pathovar, if possible. Exclude genomes should represent a diversity of strains that are closely related to, but not within, the target group, or strains that are likely to occur in the same ecological niche as the target strain. We recommend using high-quality genomes from validated members of the target groups whenever possible. 1. At the NCBI website ( select Assembly from the left dropdown menu. In the blank box on the right, enter the species or strain name of the organism you would like to target. This can optionally be followed by the operator [orgn] to ensure that only members of that organism group are returned. Click the blue Search button. Uniqprimer Tutorial 1

2 2. A page of available genome assemblies for that organism group will appear. Check the boxes next to the genomes you would like to target. You can modify the number, details, and order of the results using the options in blue letters above the Download Assemblies button. If you click no boxes, all assemblies shown will be downloaded. Select strains that you are sure are in your target group (for example, validated pathogenic strains, or strains that are not species outliers). Click the Download Assemblies box, then choose GenBank from the top dropdown box and Genomic FASTA from the bottom box. Click the Download button. Uniqprimer Tutorial 2

3 3. The FASTA-formatted genomes will be downloaded into a compressed file titled genome_assemblies.tar. The downloaded folder and sequence files need to be decompressed into regular FASTA files (ending in.fna or.fasta). The program 7-Zip can be used to extract files in Windows ( 4. Save all the include (diagnostic target) genomes to a clearly labeled folder. Download exclude genomes (closely related non-target strains) and save to a separate folder. Uniqprimer Tutorial 3

4 Part II. Uploading genomes for primer design into RiceGalaxy. 1. Navigate to the website galaxy.irri.org to open RiceGalaxy, the IRRI Galaxy instance. The Tools menu is on the left. Click the Get Data button at the top of this menu in order to upload genomes. Then, click the top link, Upload File from your computer. 2. A window will pop up asking you to select the files to upload. You can select choose local file to browse for the files, or you can drag and drop them. If your genomes are on an FTP site, you can upload them directly using the Paste/Fetch Data option. Uniqprimer Tutorial 4

5 3. Select or drag and drop the files you would like to upload. It may be easier if you upload only the exclude or nontarget genomes at this point. If you upload both target and non-target genomes, be sure they are clearly labeled so that you can tell which ones are which. 4. Click the Start button to upload the files. When all the files are 100% uploaded (they will turn green), click the Close button. The uploaded files should be listed on the right side of your Galaxy homepage. Uniqprimer Tutorial 5

6 5. Making a single combined file of exclude genomes. Under Text Manipulation Tools in the left column tools menu, click on Concatenate datasets tail-to-head. 6. Under the dropdown menu, select a file corresponding to one of the exclude genome.fna files. Click + Insert Dataset. Under the new dropdown menu that appears select another exclude genome. Repeat until all the exclude genomes are selected. Click the blue Execute button. Uniqprimer Tutorial 6

7 7. A file will be created titled Concatenate Datasets on [numbers]. You can rename it Exclude genomes by clicking on the pencil icon (Edit attributes), preview or save the file by clicking on the eye icon (View data). 8. If you haven t already done this, upload the include or diagnostic target genomes as individual files- do not concatenate these. Uniqprimer Tutorial 7

8 Part III. Generating diagnostic primers using RiceGalaxy. 1. Click on the Uniqprimer menu item under RiceGalaxy Workflow Tools in the tools column on the left. Click again on the Uniqprimer link that appears below. 2. In the dropdown menu under the FASTA file to include heading, select a.fna file that has a diagnostic target genome assembly. If there are additional diagnostic target genomes, click the button that reads + Insert Include FASTA file to generate a new dropdown box, then use this to select another include.fna file. Repeat this until you have selected all of the diagnostic target genomes as include files. 3. In the dropdown menu under the FASTA file to exclude heading, select the file that contains your exclude genomes. Uniqprimer Tutorial 8

9 4. Once your genomes are uploaded, select the size range of PCR products you would like the diagnostic primers to amplify. You can also adjust the optimal, minimal, and maximum primer sizes. It is strongly recommended to cross-validate primers for extra certainty. Click the blue Execute button. The program will take from several minutes to several hours, depending on the volume of genomes analyzed and server traffic. Three files will appear in the right-hand History column when the program is complete. In addition to a primer list, the output will also include a multifasta file that was used as the template for Primer3. The multifasta file should contain regions that are relatively distinct between include and exclude genomes, as determined by MUMmer, and can be used for other types of primer design. Finally, a Logfile will be generated that may be useful for troubleshooting and optimization. Primers can be viewed and downloaded by clicking on the eye icon next to Primer list. Uniqprimer Tutorial 9

10 Part IV. Checking primer specificity using PrimerBlast. Uniqprimer found 5 candidate primer sets that distinguish Dickeya dadantii genomes from other species of Dickeya. To determine whether these primers might result in off-target amplification of other bacterial species, we will compare them to the Genbank nr database using the program PrimerBlast. 1. Navigate to Leave the top section PCR Template blank. Under the section that says Primer Parameters, paste in the forward and reverse primers from one of the primer sets generated by Uniqprimer. Uniqprimer Tutorial 10

11 2. Scroll down to the section under the heading Primer Pair Specificity Checking Parameters. Under the Database dropdown menu, select nr to search the Genbank nr database. Alternately, you may wish to search the Refseq representative genomes database. In the Organism box, type bacteria and select bacteria (taxid 2) from the autocompleted options. 3. Click Get Primers. Uniqprimer Tutorial 11

12 4. The results will show predicted PCR products from all the bacterial species in the selected database. (This will not include most draft genomes). Results should include exact matches to target species, as shown for D. dadantii here. Ideally there should be no predicted templates in other bacterial species. However, diagnostic primers might still be considered suitable if they have predicted templates with multiple sequence mismatches in nontarget species, very long expected products, and/or target very rare species not likely to be in the testing sample. Uniqprimer Tutorial 12