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Deconstructing the polymerase chain reaction: understanding and correcting bias associated with primer degeneracies and primer-template mismatches.

Green SJ, Venkatramanan R, Naqib A - PLoS ONE (2015)

Bottom Line: The PEX PCR method substantially and significantly improved the evenness of recovery of sequences from a mock community of known composition, and allowed for amplification of templates with introduced mismatches near the 3' end of the primer annealing sites.When the PEX PCR method was applied to genomic DNA extracted from complex environmental samples, a significant shift in the observed microbial community was detected.The PEX PCR method is simple to perform, is limited to PCR mixes and a single exonuclease step which can be performed without reaction cleanup, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.

View Article: PubMed Central - PubMed

Affiliation: DNA Services Facility, Research Resources Center, University of Illinois at Chicago, Chicago, Illinois, United States of America; Dept. of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, United States of America.

ABSTRACT
The polymerase chain reaction (PCR) is sensitive to mismatches between primer and template, and mismatches can lead to inefficient amplification of targeted regions of DNA template. In PCRs in which a degenerate primer pool is employed, each primer can behave differently. Therefore, inefficiencies due to different primer melting temperatures within a degenerate primer pool, in addition to mismatches between primer binding sites and primers, can lead to a distortion of the true relative abundance of targets in the original DNA pool. A theoretical analysis indicated that a combination of primer-template and primer-amplicon interactions during PCR cycles 3-12 is potentially responsible for this distortion. To test this hypothesis, we developed a novel amplification strategy, entitled "Polymerase-exonuclease (PEX) PCR", in which primer-template interactions and primer-amplicon interactions are separated. The PEX PCR method substantially and significantly improved the evenness of recovery of sequences from a mock community of known composition, and allowed for amplification of templates with introduced mismatches near the 3' end of the primer annealing sites. When the PEX PCR method was applied to genomic DNA extracted from complex environmental samples, a significant shift in the observed microbial community was detected. Furthermore, the PEX PCR method provides a mechanism to identify which primers in a primer pool are annealing to target gDNA. Primer utilization patterns revealed that at high annealing temperatures in the PEX PCR method, perfect match annealing predominates, while at lower annealing temperatures, primers with up to four mismatches with templates can contribute substantially to amplification. The PEX PCR method is simple to perform, is limited to PCR mixes and a single exonuclease step which can be performed without reaction cleanup, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.

No MeSH data available.


Related in: MedlinePlus

Effect of PEX PCR and exonuclease treatment on observed microbial community structure and primer utilization patterns.Non-metric multidimensional scaling (NMDS) plot of lake sediment microbiome, performed at the taxonomic level of family and based on Bray-Curtis similarity (2D stress = 0.02). Samples were rarefied to 35,500 sequences per sample and no transformation was applied. All reactions were performed with an annealing temperature of 45°C, using PEX PCR with exonuclease (squares), PEX PCR without exonuclease (circles), and TAS PCR (triangles). Symbols are color-coded by the diversity (Shannon Index) of reverse primers (i.e. 806R) detected in the sequences. Maximum possible Shannon index for 18 primers in the primer pool is 2.89. Small, but significant differences in the observed family-level Shannon index (F-SI) were observed between PEX PCRs (with and without exonuclease treatment) and TAS PCRs using a two-tailed ttest (p<0.05).
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pone.0128122.g006: Effect of PEX PCR and exonuclease treatment on observed microbial community structure and primer utilization patterns.Non-metric multidimensional scaling (NMDS) plot of lake sediment microbiome, performed at the taxonomic level of family and based on Bray-Curtis similarity (2D stress = 0.02). Samples were rarefied to 35,500 sequences per sample and no transformation was applied. All reactions were performed with an annealing temperature of 45°C, using PEX PCR with exonuclease (squares), PEX PCR without exonuclease (circles), and TAS PCR (triangles). Symbols are color-coded by the diversity (Shannon Index) of reverse primers (i.e. 806R) detected in the sequences. Maximum possible Shannon index for 18 primers in the primer pool is 2.89. Small, but significant differences in the observed family-level Shannon index (F-SI) were observed between PEX PCRs (with and without exonuclease treatment) and TAS PCRs using a two-tailed ttest (p<0.05).

Mentions: Genomic DNA extracted from sediment from Lake Huron was recovered and analyzed using the TAS PCR approach, PEX PCR method and PEX PCR method without exonuclease (Fig 6). In addition, the effect of primers containing 5-nitroindole substitutions was also examined (S2 Fig). All reactions were performed with an annealing temperature of 45°C and sequencing was performed on the Illumina MiSeq instrument. Bacterial SSU rRNA amplicon sequences were clustered and analyzed at the taxonomic level of family. A significant effect of method (TAS vs PEX PCR method) was observed (ANOSIM, R = 0.778, p<0.02), as well as a significant effect of reverse primer pool (806R vs 806R-NI; R = 0.763, p<0.0003). The observed sediment sample community differed when processed using the PEX PCR method with or without exonuclease (across both regular and nitroindole primers), though analysis of similarity was not significant (R = 0.178; p<0.07). Calculation of Shannon indices for the “Sed” sample indicated that the family diversity observed in PEX PCR amplification was slightly, but significantly, higher than that observed for TAS amplification (Fig 6). Replicates of lake sediment microbial communities in analyses with the 806R-NI primer showed poorer amplification and much greater variation than replicates from the same sample with the standard 806R primer pool (S2 Fig).


Deconstructing the polymerase chain reaction: understanding and correcting bias associated with primer degeneracies and primer-template mismatches.

Green SJ, Venkatramanan R, Naqib A - PLoS ONE (2015)

Effect of PEX PCR and exonuclease treatment on observed microbial community structure and primer utilization patterns.Non-metric multidimensional scaling (NMDS) plot of lake sediment microbiome, performed at the taxonomic level of family and based on Bray-Curtis similarity (2D stress = 0.02). Samples were rarefied to 35,500 sequences per sample and no transformation was applied. All reactions were performed with an annealing temperature of 45°C, using PEX PCR with exonuclease (squares), PEX PCR without exonuclease (circles), and TAS PCR (triangles). Symbols are color-coded by the diversity (Shannon Index) of reverse primers (i.e. 806R) detected in the sequences. Maximum possible Shannon index for 18 primers in the primer pool is 2.89. Small, but significant differences in the observed family-level Shannon index (F-SI) were observed between PEX PCRs (with and without exonuclease treatment) and TAS PCRs using a two-tailed ttest (p<0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440812&req=5

pone.0128122.g006: Effect of PEX PCR and exonuclease treatment on observed microbial community structure and primer utilization patterns.Non-metric multidimensional scaling (NMDS) plot of lake sediment microbiome, performed at the taxonomic level of family and based on Bray-Curtis similarity (2D stress = 0.02). Samples were rarefied to 35,500 sequences per sample and no transformation was applied. All reactions were performed with an annealing temperature of 45°C, using PEX PCR with exonuclease (squares), PEX PCR without exonuclease (circles), and TAS PCR (triangles). Symbols are color-coded by the diversity (Shannon Index) of reverse primers (i.e. 806R) detected in the sequences. Maximum possible Shannon index for 18 primers in the primer pool is 2.89. Small, but significant differences in the observed family-level Shannon index (F-SI) were observed between PEX PCRs (with and without exonuclease treatment) and TAS PCRs using a two-tailed ttest (p<0.05).
Mentions: Genomic DNA extracted from sediment from Lake Huron was recovered and analyzed using the TAS PCR approach, PEX PCR method and PEX PCR method without exonuclease (Fig 6). In addition, the effect of primers containing 5-nitroindole substitutions was also examined (S2 Fig). All reactions were performed with an annealing temperature of 45°C and sequencing was performed on the Illumina MiSeq instrument. Bacterial SSU rRNA amplicon sequences were clustered and analyzed at the taxonomic level of family. A significant effect of method (TAS vs PEX PCR method) was observed (ANOSIM, R = 0.778, p<0.02), as well as a significant effect of reverse primer pool (806R vs 806R-NI; R = 0.763, p<0.0003). The observed sediment sample community differed when processed using the PEX PCR method with or without exonuclease (across both regular and nitroindole primers), though analysis of similarity was not significant (R = 0.178; p<0.07). Calculation of Shannon indices for the “Sed” sample indicated that the family diversity observed in PEX PCR amplification was slightly, but significantly, higher than that observed for TAS amplification (Fig 6). Replicates of lake sediment microbial communities in analyses with the 806R-NI primer showed poorer amplification and much greater variation than replicates from the same sample with the standard 806R primer pool (S2 Fig).

Bottom Line: The PEX PCR method substantially and significantly improved the evenness of recovery of sequences from a mock community of known composition, and allowed for amplification of templates with introduced mismatches near the 3' end of the primer annealing sites.When the PEX PCR method was applied to genomic DNA extracted from complex environmental samples, a significant shift in the observed microbial community was detected.The PEX PCR method is simple to perform, is limited to PCR mixes and a single exonuclease step which can be performed without reaction cleanup, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.

View Article: PubMed Central - PubMed

Affiliation: DNA Services Facility, Research Resources Center, University of Illinois at Chicago, Chicago, Illinois, United States of America; Dept. of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, United States of America.

ABSTRACT
The polymerase chain reaction (PCR) is sensitive to mismatches between primer and template, and mismatches can lead to inefficient amplification of targeted regions of DNA template. In PCRs in which a degenerate primer pool is employed, each primer can behave differently. Therefore, inefficiencies due to different primer melting temperatures within a degenerate primer pool, in addition to mismatches between primer binding sites and primers, can lead to a distortion of the true relative abundance of targets in the original DNA pool. A theoretical analysis indicated that a combination of primer-template and primer-amplicon interactions during PCR cycles 3-12 is potentially responsible for this distortion. To test this hypothesis, we developed a novel amplification strategy, entitled "Polymerase-exonuclease (PEX) PCR", in which primer-template interactions and primer-amplicon interactions are separated. The PEX PCR method substantially and significantly improved the evenness of recovery of sequences from a mock community of known composition, and allowed for amplification of templates with introduced mismatches near the 3' end of the primer annealing sites. When the PEX PCR method was applied to genomic DNA extracted from complex environmental samples, a significant shift in the observed microbial community was detected. Furthermore, the PEX PCR method provides a mechanism to identify which primers in a primer pool are annealing to target gDNA. Primer utilization patterns revealed that at high annealing temperatures in the PEX PCR method, perfect match annealing predominates, while at lower annealing temperatures, primers with up to four mismatches with templates can contribute substantially to amplification. The PEX PCR method is simple to perform, is limited to PCR mixes and a single exonuclease step which can be performed without reaction cleanup, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.

No MeSH data available.


Related in: MedlinePlus