6 - Mixed-template PCR is different than single template PCR and should be treated as such.

Just as the strategy used for single template PCRs can be optimized to account for problems such as GC-content or the presence of PCR inhibitors, so too can mixed template PCRs be optimized to account for problems like the generation of PCR artefacts.

Thoughts to consider before designing your mixed-template PCR strategy:

How can you help to preserve template to PCR product ratios?

1. Start with high template concentrations (Chandler et al., 1997; Polz and Cavanaugh, 1998).

2. Pool multiple PCRs prior to sequencing to minimize "PCR drift" (Polz and Cavanaugh, 1998; Acinas et al., 2005).

3. Keep the PCR cycle number low to minimize DNA polymerase errors and primer limitation (and chimeras, below) (Suzuki and Giovannoni, 1996; Polz and Cavanaugh, 1998; Thompson et al., 2002; Acinas et al., 2005). In this case, low means ~20 cycles (or less), or so that when product is loaded on a gel the band is barely seen.

4. Do the above, then keep in mind that PCR exponentially amplifies template from one cycle to the next. It is doubtful that you will preserve exact template to product ratios because of differences among template G+C content and primer affinity/competition, but it is reasonable to try to reduce bias wherever possible. Note, that the effect of DNA polymerase errors (and other types of error too) can also be constrained during the QC processing of raw sequences by clustering at 99% sequence similarity (Acinas et al., 2005).

How can you reduce the generation of PCR recombinants (chimeric sequences)?

1. Increase the extension time during PCR (but not excessively) (Qiu et al., 2001; Acinas et al., 2005).

2. Consider doing a 'reconditioning PCR' after your initial mixed-template PCR (Thompson et al., 2002; Acinas et al., 2005).

3. Keep the PCR cycle number low to prevent PCR artefacts from being re-amplified from cycle to cycle (Wang and Wang, 1996; Zylstra et al., 1998; Qiu et al., 2001).

4. Do the above, then be sure to screen for chimeras in your raw sequences. Chimera-calling is somewhat subjective. Chimeras made up of partial sequences from two closely related species may not be detectable. Chimeras where only a short portion of the sequence comes from another species may also not be detectable. Nevertheless, chimeras comprised of longer sequences from divergent species may be detected using existing chimera-detection tools such as those available in USEARCH/UCHIME (Edgar, 2011) or MOTHUR (Schloss et al., 2009). These tools may not catch all chimeras, they may even remove a few divergent sequences that aren't chimeras, but removing sequences of dubious quality is an important part of raw sequence clean-up.

How can you maximize the diversity of the product sequenced?

1. Consider setting up different PCR reactions, each with different sets of primers to try to avoid primer-bias (Suzuki and Giovannoni, 1996; Bellemain et al., 2010).

2. Also consider pooling multiple environmental samples (soil, water, etc.) or DNA extracts prior to PCR. Another option is to tag your samples and pool them after sequencing so that you can look at variation among your samples.

Pet-peeve: Just because some labs perform mixed-template PCR one way, doesn't necessarily make it the best way. Just because a method is widely used, doesn't make it right either. Read the literature, yes even pre-next gen literature, and learn from the pioneers before you decide on a mixed-template PCR strategy.

References:

**Acinas SG, Sarma-Rupavtarm R, Klepac-Ceraj V, Polz MF (2005) PCR-induced sequence artifacts and bias: Insights from comparison of two 16S rRNA clone libraries constructed from the same sample. Applied and Environmental Microbiology, 71:8966-8969. This paper provides a clear summary of PCR conditions to consider.

**Bik HM, Porazinska DL, Creer S, Caporaso JG, Knight R, Thomas WK (2012) Sequencing our way towards understanding global eukaryotic biodiversity. Trends in Ecology and Evolution, 27: 233-243. This paper reviews issues related to PCR generated chimeras as well as many other issues related to environmental DNA sequence analysis.

Chandler DP, Fredrickson JK, Brockman FJ (1997) Effect of PCR template concentration on the composition and distribution of total community 16S rDNA clone libraries. Molecular Ecology, 6: 475-482.

Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R (2011) UCHIME improves sensitivity and speed of chimera detection. Bioinformatics, 27: 2194-2200.

Polz MF, Cavanaugh CM (1998) Bias in template-to-product ratios in multitemplate PCR. Applied and Environmental Microbiology, 64: 3724-3730.

Qiu X, Wu L, Huang H, McDonel PE, Palumbo AV, Tiedje JM, Zhou J (2001) Evaluation of PCR-generated chimeras, mutations, and heteroduplexes with 16S rRNA gene-based cloning. Applied and Environmental Microbiology, 67: 880-887.

Schloss PD, Westcott SL, Tyabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF. Introducing mothur: Open-source, platform-independent, community-supported software for describing and comparing microbial communities. Applied and Environmental Microbiology, 75: 7537-7541.

Suzuki MT, Giovannoni SJ (1996) Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Applied and Environmental Microbiology, 62: 625-630.

Thompson JR, Marcelino LA, Polz MF (2002) Heterduplexes in mixed-template amplifications: formation, consequence and elimination by 'reconditioning PCR'. Nucleic Acids Research, 30: 2083-2088.

Wang GCY, Wang Y (1996) The frequency of chimeric molecules as a consequence of PCR co-amplification of 16S rRNA genes from different bacterial species. Microbiology, 142: 1107-1114.

Zylstra P, Rothenfluh HS, Weiller GF, Blanden RV, Steele EJ (1998) PCR amplification of murine immunoglobulin germline V genes: Strategies for minimization of recombination artefacts. Immunology and Cell Biology, 76: 395-405.