Part 6 - DNA Sequence Analysis

Sample A - Fluid from Lymph Node
(See Samples section for more info)

After the sequence information has been gathered from all the reaction tubes, the computer builds the actual sequence by matching together different pieces. You now have the 16S rDNA sequence for this bacterial species, which can be compared with all other known 16S rDNA sequences for identification.

Learn about the science behind sequence matching.

There are many sequence databases in existence. Some databases are sold commercially as part of an identification kit. In this example, we will use the GenBank public database available through the National Library of Medicine. The matching algorithm is known as BLAST (Basic Local Alignment Search Tool). A direct match of the DNA sequence determines the exact bacterial species you have found. When the DNA sequence is not an exact match but a close match to another found in the sequence database, you need to assess whether it is a new species or a variation of an existing one.

Learn more about BLAST search results.

Now follow these steps to identify Sample A:

  1. Open the data output from the sequencer. (This will open a new browser window.) Click the "Copy Sequence" button to copy the sequence data displayed inside the box.
  2. Go to the NCBI website to perform your search. (This will open a new browser window.)
  3. On the NCBI website's BLAST page, under the "Enter Query Sequence" section, paste your sequence data from step 1 into the box labeled "Enter accession number(s), gi(s), or FASTA sequence(s)." Under the "Choose Search Set: Database" section, "Standard databases (nr etc.)" and "Nucleotide collection (nr/nt)" should already be selected. When you are ready, click on the blue "BLAST" button further down the page. (Updated 4/29/2020)
  4. Follow the instructions provided by the NCBI site to obtain your results.
  5. Come back to this page (which should remain open) and identify the bacteria in the lab window to the left.