Tagged Highly Degenerate Primer (THDP)-PCR for Community Analysis of Methane- and Ammonia-oxidizing Bacteria Based on Copper-containing Membrane-bound Monooxygenases (CuMMO)    

Materials and Reagents

1. Pipette tips (20 μl, 200 μl, 1,000 μl) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog numbers: 3521-HR , 3551-HR , 3101-05-HR )
   
2. 1.5 ml Eppendorf tubes (Eppendorf, catalog number: T9661-1000EA )
   
3. Strip PCR tubes and caps (Roche Diagnostics, catalog number: 1667009001 )
   
4. PowerSoil^TM DNA Isolation Kit (Mo Bio Laboratories, catalog number: 12888-100 )
   
5. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
   
6. Premix Taq (Takara Bio, catalog number: R004A )
   
7. dNTP mixture
   
8. Tris-HCl (pH 8.3)
   
9. Potassium chloride (KCl)
   
10. Magnesium chloride (MgCl₂)
    
11. AxyPrep^TM PCR Cleanup Kit (Corning, Axygen^®, catalog number: AP-PCR-250 )
    
12. Primers
     
    

Equipment

1. Pipettes (0-10 μl, 10-100 μl, 100-1,000 μl) (Eppendorf, catalog numbers: k03030 , k03031 , k03032 )
   
2. Vortex-genie^TM 2 (Mo Bio Laboratories, model: Vortex-Genie^® 2 )
   
3. PICO 17 centrifuge (Thermo Fisher Scientific, Thermo Scientific^TM, model: Heraeus^TM Pico^TM 17 )
   
4. Automated thermal cycler TP600 (Takara Bio, model: TP600 )
   

Software

1. Mothur (www.mothur.org)
   
2. BLAST+ (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/)
   
3. Megan5 (http://ab.inf.uni-tuebingen.de/software/megan5/)

Procedure

1. Sample collection and nucleic acid extraction
   Extract genomic DNA from soil or sediment samples (0.25 g wet weight) using a PowerSoil^TM DNA Isolation Kit (Mo Bio Laboratories, USA) according to the manufacturer’s instructions.
   
2. THDP-PCR method (Figure 1)
   
   
   Figure 1. Principle of THDP-PCR
   
   
   1. First step of THDP-PCR
   1. PCR reaction recipe. Final volume: 50 μl

      H2O	18 μl
      A189-tag (10 μM)	2 μl
      HD616-tag (10 μM)	2 μl
      BSA (10 mg/ml)	2 μl
      Sample DNA	1 μl

      Note: Approximately 2 to 20 ng per sample, H₂O for negative control.
      Premix Taq (see Table 1)  25 μl
      
      Table 1. Composition of Premix Taq^TM solution
      
      
      Note: Reverse primer HD616-tag was designed based on the CuMMO sequences downloaded from FunGene Pipeline. We aligned the sequences and identified the conserved regions, then introduced degeneracies to cover different types of CuMMO gene sequences, developing the reverse primer HD616. A 20-nt-long tag was added to the 5’ end of both primers, A189 and HD616, to generate the tagged primers A189-tag and HD616-tag, respectively, for THDP-PCR amplification. Tag sequence was from virus genome (Canine astrovirus strain HUN/2012/115, GenBank: KX599351.1) and will not bind to bacterial genomes and thus avoids non-specific PCR amplification. Because tag is used as a single primer in the second step of the THDP-PCR, its length is set to 20 nt, a standard length for primers.
      
   2. Automated thermal cycler program
      1) Denaturation at 94 °C for 5 min
      2) 10 cycles at:
          94 °C for 1 min
          59 °C for 1 min
          72 °C for 1 min
      3) 72 °C for 10 min
      

      Note: Conventional (one step) PCR efficiency is not satisfactory for highly degenerate primers with degeneracy higher than 48 based on our experiment.

   2. PCR clean-up
      Purify the entire volume (50 μl) of the first-step PCR product using an AxyPrep^TM PCR Cleanup Kit (Axygen, China) according to the manufacturer’s instructions to remove unbound degenerate primers.
      Note: Unbound long tagged 3’-end unmatched degenerate primers will also bind to templates but not trigger the polymerization and thereby prevent the binding of the single tag primer to the first-step PCR products, so PCR clean-up between the two steps is necessary. 
      
   3. Second step of THDP-PCR
      
      1. PCR reaction recipe. Final volume: 50 μl
         H₂O                                                  17-19 μl
         Tag-barcode (10 μM)                       4 μl
         PCR clean-up product                      2-4 μl
         Note: Including the negative-control sample.
         Premix Taq (see Table 1)                 25 μl
         
      2. Automated thermal cycler program
         1) Denaturation at 94 °C for 5 min
         2) 35-40 cycles at:
             94 °C for 1 min
             59 °C for 1 min
             72 °C for 1 min
         3) 72 °C for 10 min
      Note: The used clean-up product volume and cycle numbers in the second step PCR can be adjusted to suit different kinds of samples. For some low-abundance samples, try to increase the clean-up product volume to 4 μl and cycle numbers to 40.
3. Pyrosequencing
   Mix together THDP-PCR products from different samples and sequence via Roche_454 pyrosequencing (GS FLX Titanium System) according to the manufacturer’s instructions.
   

Data analysis

1. Trim pyrosequencing reads using the command trim.seqs in mothur with length > 300 and q > 25, and no ambiguous sequences allowed.
   
2. Translate the trimmed sequences into amino acid sequences using FrameBot tools in Functional Gene Pipeline/Repository (http://fungene.cme.msu.edu/) to remove potential non-specific sequences which cannot be translated into CuMMO-related amino acid sequences.
   Note: The nucleotide sequences which can pass the translation are retrieved using the command get.seqs in mothur from trimmed sequences, based on this step’s results. The following analysis steps are based on the retrieved nucleotide sequences, not polypeptides.
   
3. Identify and remove chimeras using the commands chimera.uchime and remove.seqs in mothur.
   
4. Split the sequences into different samples by the barcode sequence using the command split.seqs in mothur.
   
5. Classify the split sequences with BLAST and the lowest common ancestor method in MEGAN using the expanded CuMMO database.
   Note: The original database and parameters used in this step are interpreted by Dumont et al. (2014) in the article and supplementary files. The reference database used in the classification is expanded by Wang et al. (2017) from the original database to suit the broad coverage of the highly degenerate primers, and the detailed information is listed in the article and supplementary files (Wang et al., 2017).
   

Acknowledgments

This protocol was adapted from our previous studies (Wang et al., 2017). This work was supported by the National Natural Science Foundation of China (NSFC) [grant numbers 31470222, 31170114].

References

1. Dumont, M. G., Luke, C., Deng, Y. C. and Frenzel, P. (2014). Classification of pmoA amplicon pyrosequences using BLAST and the lowest common ancestor method in MEGAN. Front Microbiol 5: 34.
   
2. Ledeker, B. M. and De Long, S. K. (2013). The effect of multiple primer-template mismatches on quantitative PCR accuracy and development of a multi-primer set assay for accurate quantification of pcrA gene sequence variants. J Microbiol Methods 94(3): 224-231.
   
3. Tuomivirta, T. T., Yrjala, K. and Fritze, H. (2009). Quantitative PCR of pmoA using a novel reverse primer correlates with potential methane oxidation in Finnish fen. Res Microbiol 160(10): 751-756.
   
4. Wang, J. G., Xia, F., Zeleke, J., Zou, B., Rhee, S. K. and Quan, Z. X. (2017). An improved protocol with a highly degenerate primer targeting copper-containing membrane-bound monooxygenase genes for community analysis of methane- and ammonia-oxidizing bacteria. FEMS Microbiol Ecol 93(3): fiw244.