Conor Meehan

16S rRNA gene homology searching using BLAST on the NCBI website

  • In this worksheet you will learn how to assign a putative species label to a 16S rRNA gene sequence in the NCBI database using the BLAST tool.

Dataset

Steps

  1. In your web browser, navigate to https://blast.ncbi.nlm.nih.gov/Blast.cgi
    • This is the homepage for BLAST, which take a query sequence (or sequences) and searches the different databases on NCBI for matches
    • See the Biological Databases and BLAST tutorial slides for a description of the different types of BLAST searches
  2. We have a nucleotide sequence and wish to compare it to other nucleotide sequences in the NCBI database.
  3. To do this, click on the ‘Nucleotide BLAST’ icon
  4. We wish to use the sequence in the file BLAST_sequence.fasta as the query so click ‘choose file’ in the ‘Enter query sequence’ section and choose that file
    • You may enter an informative title in the ‘Job title’ box if you wish
    • You can also search using an accession code from the NCBI website, which will then automatically retrieve the sequence from the database. See the ‘Searching the NCBI website for sequence data’ worksheet in the Biological Databases and BLAST tutorial for instructions on how to get this code.
  5. We will search against the entirety of the NCBI database so will leave the ‘choose search set’ database as ‘standard databases’
    • If we wished to search only within a certain species, we would put that species name in the ‘organism’ box. Start typing the name and it will bring up a list of options until you find the one you want.
      • You can also search at higher taxonomic levels such as genus or family
    • If we wish to search the whole database except for a single species/genus etc we do the same as in step 5.a but then tick the ‘exclude’ box
    • For this example we will not choose to include or exclude any specific species so leave that box blank
  6. Under ‘program selection’ we will use the standard default megablast as this is best for finding closely related homologous sequences
  7. Click the ‘BLAST’ button at the bottom of the page to start the search
  8. The results page will appear once the search is finished. This page shows you al the hits in the database that has a close match to the query sequence
  9. The primary outputs to note are:
    • The RID at the top of the page is the code needed to retrieve these results on the BLAST website if you need them in the future
    • The query length tells you the length of the sequence you input as a query
    • The ‘filter results’ section allows you to search for specific organisms or exclude other organisms to better explore the data. This is done in the same way as outlined in step 5.
    • The top 100 hits are shown in the bottom part of the page
      • Note there could be more hits than this which are hidden due to the maximum number returned being 100. Use the filter results section to better explore matches beyond the top 100
  10. For species assignment, we will use a 98% identity cut-off. Any matches over this are likely to be the same species as the query, or closely related.
    • Ideally we want a 100% match for species assignment
  11. In the ‘Filter Results’ area, set the Percent Identity to be between 98 and 100 and click ‘Filter’
    • What species remains as a hit?
    • What is the Percent identity of this hit?
  12. To ensure we dont have erroneous other potential hits. Filter the results to exclude that top hit species.
  13. Type the top hit species name into the ‘Organism’ box in the ‘Filter Results’ section. Tick the ‘exclude’ button and click ‘Filter’
    • Ensure your percent identity filters are still active
    • Do any hits to other species remain? If not, then we have a high chance that it is the species found in step 10
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