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

Materials

  • 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
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
DNA template 5 uL ~150 ng template (< 250 ng)
Nuclease-free water 19.6 uL NA
Total Volume 50 uL  
  • Place in thermocycler and run the following program under SANGER folder -> 16SFULL (bold indicates 30 cycles)
Temp (C) Time (min:sec)
95 2:00
95 0:30
55 0:30
72 1:40
72 5:00
12 hold

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 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.

  • 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.

  • Air-dry on the magnetic stand for 15 minutes.

  • Remove from the magnetic stand.

  • Add 52.5 μl RSB to each well.

  • Pipette to mix well.

  • 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

Tags: microbiome