Identifying bacterial isolates using 16S rDNA sequences:
16S rDNA sequences are very useful for identifying bacteria because they are present in all
bacterial species. However, high similarity between sequences means that it can be impossible
to distinguish between strains in the same species or family. We will use BLAST to search the
NCBI database to find the best match for 16S rDNA sequences from our unknown isolates.
Steps to identify bacteria using 16S rRNA genes:
1) DNA purification from cultures of bacteria
2) PCR of 16s rRNA gene using “universal” bacteria primers
3) Clean-up of PCR samples to remove primers and dNTPs
4) Cycle sequencing of 16S rRNA gene
5) Comparison of sequenced 16S rRNA gene to known sequences
Trace data:
After sequencing the DNA, the sequencing facility sends back a file with trace data and a file
with a DNA sequence.
1. Go the class website. Download the documents for this lab.
2. Take a look at Example 1 of trace data on page 4. Notice that there are four colors of
peaks representing the four DNA nucleotides (see version on your screen for colors).
The beginning and ends of the sequences are often of poor quality and must be cropped
from sequences. Sometimes the computer program can’t determine the correct base at
a position and you need to fix it.
3. Use the colors of the peaks to fill in the blanks. (Write your answer on #1 in the
Questions)
______
4. We also checked the quality of the peaks. Sometimes there are overlapping peaks,
suggesting that there may be multiple sequences. Example 2 on page 4 shows this. The
computer program used the tallest peak at each position to select the base.
Procedure for searching database:
1. Go the class website. Download the documents with the rDNA gene sequences. Then go
to the NCBI BLAST website:
http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_L
OC=blasthome
(Or search for NCBI BLAST, selecting Nucleotide Blast)
2. Copy and paste your 16S rDNA sequences, one at a time, from the word document into
the “Enter Query Sequence Box”, and then hit tab. This will enter your sequence and
title.
3. Under database, make sure “16S ribosomal RNA” is selected.
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4. In program selection, make sure you have selected “Highly Similar”, then hit BLAST.
Nucleotide BLAST window:
5. You should now have a list of possible matches for your sequence. Look at the top hits
with equivalent scores (especially E-value and Ident[ity] values) in the columns at the
right. Usually, the genera (plural for genus) will all match. If there are different genera,
you must further investigate to find the similarities.
6. If you have different genera, select the first genus and right click on the number
underneath Accession to display in new tab. Under Genera information, click the link
after “Organism”.
7. You should now have a detailed lineage highlighted in blue for that species:
Example: Escherichia coli
Lineage( full )(NCBI Taxonomy Browser)
cellular organisms; Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales;
Enterobacteriaceae; Escherichia
Superkingdom: Bacteria
Phylum: Proteobacteria
Class: Gammaproteobacteria (ɣ-proteobacteria)
Order: Enterobacteriales
Family : Enterobacteriaceae
Genus : Escherichia
Species: coli
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8. Repeat steps 6 – 8 for each of your top genera, and look at each lineage to see what
they have in common. Try to be as specific as possible.
9. Write down the most specific identification (genus or family) for your bacterial isolate
(Question #3).
10. Repeat with other sequences.
11. Once you have identified your unknown strain, find out more information about that
group of bacteria.
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Example Sequence Traces:
Example 1:
Example 2:
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Questions for 16S rDNA sequencing
1. Use the colors of the peaks for the bases to fill in the blanks (See color version on electronic
version of lab procedure):
______
2. Briefly describe the steps to sequence the DNA (starting with extraction from the samples of
bacteria):
3. After you have completed BLAST with your unknown sequences, fill in the table:
Sequence
Identification
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2
3
4
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4. Look up more information about the family/genera of bacteria you have identified.
5. Are there any strain identifications that don’t make sense? If so, explain.
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