- Before starting
- Materials
- Obtain genomic DNA
- PCR Amplify the full-length 16S rRNA gene
- Cleanup
- QC on purified PCR products
- Submit PCR products for sequencing
- Step 6: Interpret results
Before starting
- 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
- 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 |
Materials
- Promega GoTaq Green Master Mix (-20C)
- Molecular biology grade, nuclease-free water (-20C or room temperature)
- 27F Primer
- 1492R primer
- 515F primer
- Ampure XP Beads (remove from fridge and acclimate at room temp for 30 min before use)
- 100% Ethanol
Obtain genomic DNA
- Extract DNA using PureLink DNA protocol (bacterial protocol) or other method of bacterial DNA extraction.
- Quantify DNA using the Qubit.
PCR Amplify the full-length 16S rRNA gene
Prepare the following, per sample, as a master mix:
| Component | Volume | Final Concentration |
|---|---|---|
GoTaq Green Master Mix, 2X | 25 uL | 1X |
27F primer, 100 uM | 0.2 uL | 0.2 uM |
1492R primer, 100 uM | 0.2 uL | 0.2 uM |
Nuclease-free water | 19.6 uL | NA |
Total Volume | 45 uL |
Make a master mix including all of the components except for the DNA.
The original IDT stock solutions of the primers are at a concentration of 100 uM. Our primers are at this concentration in the freezer. If your primers are not this concentration, adjust the volume added.
In a 96-well plate, pipette 45uL of prepared master mix per sample.
Add 5uL of extracted DNA template (~150 ng template (< 250 ng)) and pipette to mix. Each sample will have a total volume of 50uL.
Place in thermocycler and run the following program under SANGER folder -> 16SFULL (bold indicates 30 cycles)
Cleanup
Vortex AMPure XP beads before each use. Vortex AMPure XP beads frequently to make sure that beads are evenly distributed.
Add 50 μl of AMPure XP beads to each well.
Pipette to mix around 15 times to ensure the beads are mixed well with PCR products.
Incubate at room temperature for 5 minutes.
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
Remove and discard all supernatant from each well.
Keeping the samples on the magnetic stand, wash 2 times as follows:
- Add 180 μl fresh 80% EtOH to each well.
- Incubate on the magnetic stand for 30 seconds.
- Remove and discard all supernatant from each well.
Using a 20 μl pipette, remove residual 80% EtOH from each well.
Let the plate stand on the magnet at RT until dry, usually less than 5 minutes. When dry, the beads will appear matte and cracked.
Remove from the magnetic stand when samples are dry.
Add 52.5 μl RSB to each well.
Pipette to mix well and resuspend beads.
Incubate at room temperature for 2 minutes.
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
Transfer 50 μl supernatant to a new plate.
This is a safe stopping point. If you choose to stop here, seal the plate and store at -20°C for up to 7 days. Alternatively, leave in the thermal cycler overnight.
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.
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.
We typically submit 3 tubes per sample: a 27F, 515F, and 1492R. These three sequencing results help me assemble the 16S gene without gaps.
Use strip tubes with the strip caps for easy removal if you have <52 samples. If you have more than 52 samples, submit your samples in a plate with the HT option.
The first tube of each 8-tube set should have ‘Beiting’ on it and the second tube should have your last name. Label your samples sequentially 1, 2, 3, etc….
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
No database is comprehensive and there’s always bias in what is represented in a database. In addition, since you’re only considering one gene (16S rRNA), this also affects your specificity and resolution for identifying species.