- The protocol uses three primers (two forward and one reverse) to generate amplicons for Sanger sequencing. Combining the seqeunce data for the three amplicons generates a contiguous sequence that spans that majority of the full 16S rRNA gene, therby giving you a decent idea of bacterial identity. PCR reactions use the GoTaq Master Mix
- Promega GoTaq Green Master Mix (-20C)
- Molecular biology grade, nuclease-free water (-20C or room temperature)
- 27F Primer
- 1492R primer
- 515F primer
- QIAGEN QIAquick PCR purification kit
- 100% Ethanol
Step 1: Obtain genomic DNA
- Extract DNA using QIAGEN DNeasy kit (bacterial protocol) or other method of bacterial DNA extraction.
- Quantify DNA using the Qubit.
Step 2: PCR Amplify the full-length 16S rRNA gene
- PCR Primers are as follows:
|name||Sequence (5’ -> 3’)|
|27fwd||AGA GTT TGA TCM TGG CTC AG|
|515fwd||GTG CCA GCM GCC GCG GTA A|
|1492rev||CGG TTA CCT TGT TAC GAC TT|
- Prepare the following reaction mixture in each PCR well/tube
|GoTaq Green Master Mix, 2X||25 uL||1X|
|27F primer, 10 uM||1 uL||0.2 uM|
|1492R primer, 10 uM||1 uL||0.2 uM|
|DNA template||1-5 uL||~150 ng template (< 250 ng)|
|Nuclease-free water to||50 uL||NA|
- Place in thermocycler and run the following program under CHMI -> PCR -> 16SFULL (bold indicates 30 cycles)
|Temp (C)||Time (min:sec)|
Step 3. Clean up the PCR products
- Follow the QIAGEN QIAquick PCR purification protocol.
Step 4: QC on purified PCR products
Perform Qubit quantification on all samples
Optional: Select a few samples and run an agarose gel or tapestation (DNA 5000 tape) to ensure there is a major product at approximately 1400 bp.
Step 5: Submit PCR products for sequencing
Follow the submission guide instructions carefully for submitting to the UPenn DNA sequencing facility
Each tube should contain the following:
- 6 uL of PCR product at 25 ng/uL
- 3 uL of the desired primer at 1.1 uM
- If your DNA is not sufficiently concentrated, just submit the sample anyway, your results may be fine.
- The sequencing facility suggests 10 ng of DNA per 100 bp.
Step 6: Interpret results
Assemble the sequencing reads using your favorite assembly software. We use Geneious (commercial software) because it has an easy-to-use interface. .Ab1 files for all three amplicons can be dragged onto the Geneious interface and the de novo assembly tool is used to produce a consensus contig.
Take the highest quality portion of the full length consensus sequence and seach for the best match using either BLAST or a recent tool called Bitsliced Genomic Signature Index (BIGSI) which is available here and you can read more about on the Github page for BIGSI