Identification of unknown bacterial isolates using Sanger sequencing of the 16S rRNA gene

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:

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

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

• Prepare the following, per sample, as a master mix:

• 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)

Step 3: 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.

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