BACKGROUND
in the field of microbial ecology, the polymerase chain reaction  has been widely used for the amplification, detection and quantification of dna targets since its introduction  <cit> , resulting in increased knowledge of the microbial world  <cit> .
however, the efficiency and accuracy of pcr can be diminished by many factors including primer-template mismatches, reactant concentrations, the number of pcr cycles, annealing temperature, the complexity of the dna template, and others.
<cit> .
primer-template mismatches are the most important because they can lead to selective amplification which prevents the correct assessment of microbial diversity  <cit> .
target sequences that cannot match the primers precisely will be amplified to a lesser extent, possibly even below the detection limit.
the relative content of the sequences achieved is therefore changed, resulting in a deviation from the true community composition.
hence a comprehensive evaluation of bacterial primer coverage is critical to the interpretation of pcr results in microbial ecology research.
many related studies on primer coverage have been performed previously, but most are qualitative or semi-quantitative studies restricted to the domain level  <cit> .
low coverage rates in some rare phyla might have been overlooked.
although wang et al.
<cit>  investigated primer coverage rates at the phylum level, only sequences from the ribosomal database project  were used.
this sole reliance on the rdp is another common limitation of previous studies.
the rdp is a professional database containing more than one million 16s rrna gene sequences.
it also provides a series of data analysis services  <cit> , including probe match, which is often used in primer studies.
however, despite the rdp’s large collection of sequences and extensive application, most of its sequences were generated through pcr amplification.
sequences that fail to match the universal primers may become lost in the pcr results, and so are not included in the rdp.
consequently, primer coverage rates in the rdp appear to be higher than they actually are.
fortunately, with the rapid development of sequencing techniques, many large-scale metagenomic datasets have become available.
metagenomic sequences are generated directly from sequencing environmental samples and are free of pcr bias; thus, the resulting datasets faithfully reflect microbial composition, especially in the case of rare biospheres.
the community cyberinfrastructure for advanced microbial ecology research and analysis  is not only a repository for rich and distinctive metagenomic data, but it also provides a set of bioinformatic tools for research <cit> .
another shortcoming of previous primer-coverage studies has recently been illuminated through studies on the pcr mechanism.
in the past, it was assumed that a single primer-template mismatch would not obstruct amplification under proper annealing temperature so long as the mismatch did not occur at the 3′ end of the primer.
however, recent studies have shown that a single mismatch within the last 3– <dig> nucleotides of the 3′ end could also significantly reduce pcr amplification efficiency, even under optimal annealing temperature  <cit> .
this changed the criteria for judging whether a primer binding-site sequence could be amplified faithfully by pcr.
in this study, we define sequences that “match with” the primers as having either no mismatch with the primer, or as having only one mismatch that is not located within the last  <dig> nucleotides of the 3′ end.
all of the primers in this study are frequently used in molecular microbial ecology research.
the most common primer pairs are 27f and 1390r/1492r, which are mainly used for constructing clone libraries of the full-length 16s rdna sequence  <cit> .
the primers such as 338f and 338r are frequently used in pyrosequencing  <cit> .
the remaining primers are most commonly used for fingerprint analyses, but the development of next-generation sequencing techniques will likely broaden their roles in future studies  <cit> .
pyrosequencing has extended the read length from 100bp to 800bp  <cit> , and as a result, hypervariable regions in 16s rdna other than v <dig> and v <dig> will be able to be sequenced.
those primers that can cover these hypervariable regions will become more frequently used.
the aim of this study was to assess the coverage rates of  <dig> common primers , which target different regions of the bacterial 16s rrna gene, using sequences from the rdp and  <dig> metagenomic datasets.
we used the non-coverage rate, the percentage of sequences that could not match with the primer, as the major indicator in this study.
non-coverage rates were calculated at both the domain and phylum levels, and the influence of a single mismatched position on the non-coverage rate was analyzed.
by comparing the rdp and the metagenomic datasets, we found that the non-coverage rates were seriously underestimated when only the rdp dataset was used.
RESULTS
influence of a single mismatch in the last  <dig> nucleotides
since the beginning of the 1990s, it has been widely acknowledged that pcr amplification is significantly inhibited by a single mismatch occurring at the 3′ end of the primer  <cit> .
even when the last nucleotide was substituted with inosine, which is capable of binding to all four nucleotides, primers still failed to amplify all of the expected sequences in the microbial community  <cit> .
recently, bru et al.
<cit>  and wu et al.
<cit>  demonstrated that the efficiency of pcr amplification was also inhibited if a single mismatch occurred within the last 3– <dig> nucleotides of the 3′ end of primer, even when the annealing temperature was decreased for optimal efficiency.
these single mismatches have not been considered in previous primer coverage studies  <cit> .
we studied the influence of a single primer mismatch occurring within the last  <dig> nucleotides using the rdp dataset.
at the domain level, a relatively weak influence was found when non-coverage rates that allowed a single mismatch in the last  <dig> nucleotides were compared to rates that did not allow such a mismatch.
the absolute differences were ≪5% for all of the primers except 519f .
in contrast, significant differences were observed for some of the primers at the phylum level.
rate differences ≫20% under two criteria are listed in table  <dig>  the most noticeable non-coverage rate was observed for 338f in the phylum lentisphaerae.
if a single mismatch was allowed within the last  <dig> nucleotides, its non-coverage rate was only 3%; otherwise, it was as high as 100%.
similar results were observed for 338f in the phylum op <dig>  but with a smaller number of sequences.
these results indicate that 338f is not appropriate for either phylum .
overall, the most seriously affected primer was 519f.
in this case,  <dig> phyla showed rate differences ≫20% under two criteria, and  <dig> phyla showed differences ≫40%.
the significant differences observed at the phylum level imply that a single mismatch in the last  <dig> nucleotides may be fatal under specific circumstances, and this possibility should be considered when choosing and designing primers.
at the phylum level, non-coverage rates that changed more than 20% under two criteria are listed.
“non-coverage rate 4+” denotes the non-coverage rate when a single mismatch in the last  <dig> nucleotides was allowed.
“non-coverage rate 4-” denotes the non-coverage rate when mismatches in the last  <dig> nucleotides were not allowed.
non-coverage rates of  <dig> primers at the domain level
non-coverage rates for the  <dig> common primers relative to the  <dig> datasets examined were calculated .
in the rdp dataset, the non-coverage rate for primer 27f reached  <dig> %, but the rates of the other  <dig> primers were all ≪6%.
however, in the metagenomic datasets,  <dig> out of  <dig>  non-coverage rates were ≫10%.
moreover, for all primers except 27f, the average rates from the  <dig> metagenomic datasets were at least 4-times higher than in the rdp dataset, and the ratio even reached  <dig>  for the primer 519r.
normalized results were similar .
the average difference between the rdp and the metagenomic datasets was  <dig> % before and  <dig> % after normalization.
the average absolute difference between the original and normalized domain non-coverage rates was  <dig> %.
these results revealed that the non-coverage rates in the rdp were greatly underestimated and proved the effectiveness of using metagenomes to assess primer coverage.
furthermore, after eliminating primer contamination , most of the sequences containing a 27f binding site in the rdp came from the metagenomes.
this might explain why the non-coverage rate for 27f in the rdp dataset was close to that in the metagenomic datasets.
non-coverage rates for  <dig> primers at the phylum level
because each dataset is a mixture of sequences from various microbes occurring in various proportions according to different phyla, low coverage of minor phyla could be easily masked by the higher coverage of the dominant phyla.
moreover, the compositions of microbial communities differ greatly with environments; minor microbes found in common environments may in fact be major components in other ecological niches.
it is therefore necessary to assess the non-coverage rates at the phylum level in the different metagenomic datasets.
338f and 338r
non-coverage rates for the primers 338f and 338r varied among different phyla .
in the rdp dataset, the non-coverage rates for 338f in  <dig> phyla  were ≫95%.
primer binding-site sequences that could not match with primer 338f are listed in additional file 3: table s <dig>
in the rdp dataset, the most frequent sequence variant retrieved  was 338f-3a12t .
this sequence was the major variant in the verrucomicrobia, accounting for  <dig> % of the sequences in the rdp dataset and  <dig> % in the gos  dataset; it also predominated in the phyla chloroflexi, brc <dig>  op <dig> and op <dig>  the second variant, 338f-16t, was the major variant in the lentisphaerae but also appeared in many other phyla.
the third variant, 338f-3a12t16t, was specific for planctomycetes and od <dig>  and accounted for approximately 50% of planctomycetes in both the rdp and gos datasets.
the variants 338f-4t11a and 338f-12g were distributed in various phyla, while 338f-3c12g was specific for aquificae and 338f-3c4t11a12g for cyanobacteria.
also significant was the non-coverage rate for 338f in the actinobacteria.
in the rdp dataset, this rate was only  <dig> %, but in the metagenomic datasets, the results were substantially different.
the non-coverage rates in the gos and hot datasets, for example, were  <dig> % and  <dig> %, respectively.
we observed that the absolute number of 338f-16t sequences from actinobacteria in the rdp dataset was  <dig>  which was much larger than the numbers in the gos and hot datasets.
the implication is that the 338f-16t actinobacteria sequences in the rdp most likely came from environments similar to those from which the gos and hot sequences were sampled.
for the primer 338r, the reverse complement of 338f, the homologous variants 338f-16t and 338f-16c had no effect on the non-coverage rate, while three other variants  warranted further attention .
although hundreds of sequences for each variant were found, they accounted for low percentages of the major phyla .
variants with more than one mismatch were similar to those of 338f.
the bisonmetagenome dataset was dominated by aquificae and the non-coverage rates for both 338f and 338r in aquificae were 100%.
the sequence variant 338f-3c12g  was the major type.
thus, the primers 338f/338r might not be appropriate for the analysis of hot spring samples or the detection of aquificae.
519f and 519r
the coverage of primer 519r was quite “universal” except for its high non-coverage rate in the phylum od <dig> in the antarcticaaquatic dataset, where the primer binding-site sequence variant 519r-14t-11t12c had a rate of  <dig> %.
although non-coverage rates of approximately 20% were found scattered across other phyla, these rates resulted from variants with only one or two sequences, and no dominating variant was found.
overall, primer 519r could authentically amplify sequences from most phyla.
a substantial difference was found between the non-coverage rates of 519f and 519r.
five sequence variants were mainly responsible for the high non-coverage rate for 519f .
notably, the  <dig> most dominant variants had one trait in common – a single mismatch at the 16th nucleotide .
this mismatch did not influence the non-coverage rate of 519r.
further analysis showed that the high non-coverage rate of 519f was caused primarily by sequences from the phylum nitrospirae.
the acidmine metagenome is dominated by leptospirillum species of the nitrospirae, and therefore forms an ideal dataset for nitrospirae studies  <cit> .
of the 519f-binding sequences in the dataset, 89% were from nitrospirae, and none could match with 519f.
the non-coverage rate in the rdp dataset was also high  in nitrospirae, whereas the total non-coverage rate for 519f in the rdp dataset was only 6%.
similar sample analyses should therefore be focused on the use of primer 519f.
other primers
frank et al.
<cit>  have studied the 27f and 1492r primer pair and have proposed 27f-ym +  <dig> as a modification of the common 27f primer.
our results support this modification as being necessary .
the non-coverage rates for 1390r and 1492r were quite low, even at the phylum level.
for primer 907r, only one sequence variant that could not match with the primer  was observed.
it resulted in the high non-coverage rate observed in phylum tm <dig> .
CONCLUSIONS
the 16s rrna gene is an important genetic marker for the characterization of microbial community structure by 16s rrna gene amplicon sequencing with conserved primers  <cit> .
because of the increase in read length with the development of pyrosequencing  technology, different multi-hypervariable regions can be selected for amplification.
in this strategy, different pairs of “universal” primers are used for barcoded pyrosequencing  <cit> .
however, even with pyrosequencing, the bias caused by primer-template mismatch may misrepresent the real community composition of environmental samples.
therefore, the assessment of primer coverage to perfect the use of universal primers is urgently required.
in this study, we assessed the non-coverage rates for  <dig> common universal bacterial primers in the rdp dataset and  <dig> metagenomic datasets.
comparisons of non-coverage rates, with or without constraining the position of a single mismatch, emphasized the importance of further study of the mechanism of pcr.
metagenomic dataset analysis revealed that some sequence variants, which appeared to be minor in the public databases, were actually dominant in some ecological niches.
these results are of great practical significance for studies on similar environmental samples, and new primer formulations could be designed using our results.
one strategy is to increase coverage through the introduction of proper degenerate nucleotides.
although the total number of sequences in a metagenomic dataset may be very large, the number of 16s rrna gene sequences is limited, and may account for only approximately  <dig> % of all sequence reads  <cit> .
in contrast, the metatranscriptomic analysis of environmental samples generates a large number of small subunit sequences  <cit> .
although the short length  of the sequences currently deposited in metatranscriptomic datasets are not appropriate for assessing primer coverage, the further development of pyrosequencing will make such assessments possible in the near future.

 in the field of microbial ecology, the polymerase chain reaction has been widely used for the amplification, detection and quantification of dna targets since its introduction, resulting in increased knowledge of the microbial world. however, the efficiency and accuracy of pcr can be diminished by many factors including primer-template mismatches, reactant concentrations, the number of pcr cycles, annealing temperature, the complexity of the dna template, and others.. primer-template mismatches are the most important because they can lead to selective amplification which prevents the correct assessment of microbial diversity. target sequences that cannot match the primers precisely will be amplified to a lesser extent, possibly even below the detection limit. the relative content of the sequences achieved is therefore changed, resulting in a deviation from the true community composition. hence a comprehensive evaluation of bacterial primer coverage is critical to the interpretation of pcr results in microbial ecology research. many related studies on primer coverage have been performed previously, but most are qualitative or semi-quantitative studies restricted to the domain level. low coverage rates in some rare phyla might have been overlooked. although wang et al. investigated primer coverage rates at the phylum level, only sequences from the ribosomal database project were used. this sole reliance on the rdp is another common limitation of previous studies. the rdp is a professional database containing more than one million 16s rrna gene sequences. it also provides a series of data analysis services, including probe match, which is often used in primer studies. however, despite the rdp’s large collection of sequences and extensive application, most of its sequences were generated through pcr amplification. sequences that fail to match the universal primers may become lost in the pcr results, and so are not included in the rdp. consequently, primer coverage rates in the rdp appear to be higher than they actually are. fortunately, with the rapid development of sequencing techniques, many large-scale metagenomic datasets have become available. metagenomic sequences are generated directly from sequencing environmental samples and are free of pcr bias; thus, the resulting datasets faithfully reflect microbial composition, especially in the case of rare biospheres. the community cyberinfrastructure for advanced microbial ecology research and analysis is not only a repository for rich and distinctive metagenomic data, but it also provides a set of bioinformatic tools for research. another shortcoming of previous primer-coverage studies has recently been illuminated through studies on the pcr mechanism. in the past, it was assumed that a single primer-template mismatch would not obstruct amplification under proper annealing temperature so long as the mismatch did not occur at the 3′ end of the primer. however, recent studies have shown that a single mismatch within the last 3– nucleotides of the 3′ end could also significantly reduce pcr amplification efficiency, even under optimal annealing temperature. this changed the criteria for judging whether a primer binding-site sequence could be amplified faithfully by pcr. in this study, we define sequences that “match with” the primers as having either no mismatch with the primer, or as having only one mismatch that is not located within the last nucleotides of the 3′ end. all of the primers in this study are frequently used in molecular microbial ecology research. the most common primer pairs are 27f and 1390r/1492r, which are mainly used for constructing clone libraries of the full-length 16s rdna sequence. the primers such as 338f and 338r are frequently used in pyrosequencing. the remaining primers are most commonly used for fingerprint analyses, but the development of next-generation sequencing techniques will likely broaden their roles in future studies. pyrosequencing has extended the read length from 100bp to 800bp, and as a result, hypervariable regions in 16s rdna other than v and v will be able to be sequenced. those primers that can cover these hypervariable regions will become more frequently used. the aim of this study was to assess the coverage rates of common primers, which target different regions of the bacterial 16s rrna gene, using sequences from the rdp and metagenomic datasets. we used the non-coverage rate, the percentage of sequences that could not match with the primer, as the major indicator in this study. non-coverage rates were calculated at both the domain and phylum levels, and the influence of a single mismatched position on the non-coverage rate was analyzed. by comparing the rdp and the metagenomic datasets, we found that the non-coverage rates were seriously underestimated when only the rdp dataset was used.  influence of a single mismatch in the last nucleotides since the beginning of the 1990s, it has been widely acknowledged that pcr amplification is significantly inhibited by a single mismatch occurring at the 3′ end of the primer. even when the last nucleotide was substituted with inosine, which is capable of binding to all four nucleotides, primers still failed to amplify all of the expected sequences in the microbial community. recently, bru et al. and wu et al. demonstrated that the efficiency of pcr amplification was also inhibited if a single mismatch occurred within the last 3– nucleotides of the 3′ end of primer, even when the annealing temperature was decreased for optimal efficiency. these single mismatches have not been considered in previous primer coverage studies. we studied the influence of a single primer mismatch occurring within the last nucleotides using the rdp dataset. at the domain level, a relatively weak influence was found when non-coverage rates that allowed a single mismatch in the last nucleotides were compared to rates that did not allow such a mismatch. the absolute differences were ≪5% for all of the primers except 519f. in contrast, significant differences were observed for some of the primers at the phylum level. rate differences ≫20% under two criteria are listed in table the most noticeable non-coverage rate was observed for 338f in the phylum lentisphaerae. if a single mismatch was allowed within the last nucleotides, its non-coverage rate was only 3%; otherwise, it was as high as 100%. similar results were observed for 338f in the phylum op but with a smaller number of sequences. these results indicate that 338f is not appropriate for either phylum. overall, the most seriously affected primer was 519f. in this case, phyla showed rate differences ≫20% under two criteria, and phyla showed differences ≫40%. the significant differences observed at the phylum level imply that a single mismatch in the last nucleotides may be fatal under specific circumstances, and this possibility should be considered when choosing and designing primers. at the phylum level, non-coverage rates that changed more than 20% under two criteria are listed. “non-coverage rate 4+” denotes the non-coverage rate when a single mismatch in the last nucleotides was allowed. “non-coverage rate 4-” denotes the non-coverage rate when mismatches in the last nucleotides were not allowed. non-coverage rates of primers at the domain level non-coverage rates for the common primers relative to the datasets examined were calculated. in the rdp dataset, the non-coverage rate for primer 27f reached %, but the rates of the other primers were all ≪6%. however, in the metagenomic datasets, out of non-coverage rates were ≫10%. moreover, for all primers except 27f, the average rates from the metagenomic datasets were at least 4-times higher than in the rdp dataset, and the ratio even reached for the primer 519r. normalized results were similar. the average difference between the rdp and the metagenomic datasets was % before and % after normalization. the average absolute difference between the original and normalized domain non-coverage rates was %. these results revealed that the non-coverage rates in the rdp were greatly underestimated and proved the effectiveness of using metagenomes to assess primer coverage. furthermore, after eliminating primer contamination, most of the sequences containing a 27f binding site in the rdp came from the metagenomes. this might explain why the non-coverage rate for 27f in the rdp dataset was close to that in the metagenomic datasets. non-coverage rates for primers at the phylum level because each dataset is a mixture of sequences from various microbes occurring in various proportions according to different phyla, low coverage of minor phyla could be easily masked by the higher coverage of the dominant phyla. moreover, the compositions of microbial communities differ greatly with environments; minor microbes found in common environments may in fact be major components in other ecological niches. it is therefore necessary to assess the non-coverage rates at the phylum level in the different metagenomic datasets. 338f and 338r non-coverage rates for the primers 338f and 338r varied among different phyla. in the rdp dataset, the non-coverage rates for 338f in phyla were ≫95%. primer binding-site sequences that could not match with primer 338f are listed in additional file 3: table s in the rdp dataset, the most frequent sequence variant retrieved was 338f-3a12t. this sequence was the major variant in the verrucomicrobia, accounting for % of the sequences in the rdp dataset and % in the gos dataset; it also predominated in the phyla chloroflexi, brc op and op the second variant, 338f-16t, was the major variant in the lentisphaerae but also appeared in many other phyla. the third variant, 338f-3a12t16t, was specific for planctomycetes and od and accounted for approximately 50% of planctomycetes in both the rdp and gos datasets. the variants 338f-4t11a and 338f-12g were distributed in various phyla, while 338f-3c12g was specific for aquificae and 338f-3c4t11a12g for cyanobacteria. also significant was the non-coverage rate for 338f in the actinobacteria. in the rdp dataset, this rate was only %, but in the metagenomic datasets, the results were substantially different. the non-coverage rates in the gos and hot datasets, for example, were % and %, respectively. we observed that the absolute number of 338f-16t sequences from actinobacteria in the rdp dataset was which was much larger than the numbers in the gos and hot datasets. the implication is that the 338f-16t actinobacteria sequences in the rdp most likely came from environments similar to those from which the gos and hot sequences were sampled. for the primer 338r, the reverse complement of 338f, the homologous variants 338f-16t and 338f-16c had no effect on the non-coverage rate, while three other variants warranted further attention. although hundreds of sequences for each variant were found, they accounted for low percentages of the major phyla. variants with more than one mismatch were similar to those of 338f. the bisonmetagenome dataset was dominated by aquificae and the non-coverage rates for both 338f and 338r in aquificae were 100%. the sequence variant 338f-3c12g was the major type. thus, the primers 338f/338r might not be appropriate for the analysis of hot spring samples or the detection of aquificae. 519f and 519r the coverage of primer 519r was quite “universal” except for its high non-coverage rate in the phylum od in the antarcticaaquatic dataset, where the primer binding-site sequence variant 519r-14t-11t12c had a rate of %. although non-coverage rates of approximately 20% were found scattered across other phyla, these rates resulted from variants with only one or two sequences, and no dominating variant was found. overall, primer 519r could authentically amplify sequences from most phyla. a substantial difference was found between the non-coverage rates of 519f and 519r. five sequence variants were mainly responsible for the high non-coverage rate for 519f. notably, the most dominant variants had one trait in common – a single mismatch at the 16th nucleotide. this mismatch did not influence the non-coverage rate of 519r. further analysis showed that the high non-coverage rate of 519f was caused primarily by sequences from the phylum nitrospirae. the acidmine metagenome is dominated by leptospirillum species of the nitrospirae, and therefore forms an ideal dataset for nitrospirae studies. of the 519f-binding sequences in the dataset, 89% were from nitrospirae, and none could match with 519f. the non-coverage rate in the rdp dataset was also high in nitrospirae, whereas the total non-coverage rate for 519f in the rdp dataset was only 6%. similar sample analyses should therefore be focused on the use of primer 519f. other primers frank et al. have studied the 27f and 1492r primer pair and have proposed 27f-ym +  as a modification of the common 27f primer. our results support this modification as being necessary. the non-coverage rates for 1390r and 1492r were quite low, even at the phylum level. for primer 907r, only one sequence variant that could not match with the primer was observed. it resulted in the high non-coverage rate observed in phylum tm.  the 16s rrna gene is an important genetic marker for the characterization of microbial community structure by 16s rrna gene amplicon sequencing with conserved primers. because of the increase in read length with the development of pyrosequencing technology, different multi-hypervariable regions can be selected for amplification. in this strategy, different pairs of “universal” primers are used for barcoded pyrosequencing. however, even with pyrosequencing, the bias caused by primer-template mismatch may misrepresent the real community composition of environmental samples. therefore, the assessment of primer coverage to perfect the use of universal primers is urgently required. in this study, we assessed the non-coverage rates for common universal bacterial primers in the rdp dataset and metagenomic datasets. comparisons of non-coverage rates, with or without constraining the position of a single mismatch, emphasized the importance of further study of the mechanism of pcr. metagenomic dataset analysis revealed that some sequence variants, which appeared to be minor in the public databases, were actually dominant in some ecological niches. these results are of great practical significance for studies on similar environmental samples, and new primer formulations could be designed using our results. one strategy is to increase coverage through the introduction of proper degenerate nucleotides. although the total number of sequences in a metagenomic dataset may be very large, the number of 16s rrna gene sequences is limited, and may account for only approximately % of all sequence reads. in contrast, the metatranscriptomic analysis of environmental samples generates a large number of small subunit sequences. although the short length of the sequences currently deposited in metatranscriptomic datasets are not appropriate for assessing primer coverage, the further development of pyrosequencing will make such assessments possible in the near future. 
additionally, most studies do not account for the positional effect of single primer-template mismatches. the coverage of universal primers for the bacterial 16s rrna gene plays a crucial role in the correct understanding of microbial community structure. with the development of high-throughput, next-generation sequencing techniques, it will become feasible to sequence more of the hypervariable regions of the bacterial 16s rrna gene. however, existing studies on primer coverage are limited by the lack of appropriate databases and are restricted to the domain level. in this study, we used metagenomic datasets as well as the ribosomal database project to assess the coverage of widely used bacterial primers. the coverage rates for bacterial primers were found to be overestimated by previous studies that only investigated the rdp because of pcr amplification bias in the sequence composition of the dataset. in the rdp, the non-coverage rates for all primers except 27f were ≪6%, while in the metagenomic datasets, most were ≫10%. if one considers that a single mismatch near the 3′ end of the primer might greatly reduce pcr efficiency, then some phylum non-coverage rates would change by more than 20%.
 the coverage of universal primers for the bacterial 16s rrna gene plays a crucial role in the correct understanding of microbial community structure. however, existing studies on primer coverage are limited by the lack of appropriate databases and are restricted to the domain level. additionally, most studies do not account for the positional effect of single primer-template mismatches. in this study, we used metagenomic datasets as well as the ribosomal database project to assess the coverage of widely used bacterial primers.  the coverage rates for bacterial primers were found to be overestimated by previous studies that only investigated the rdp because of pcr amplification bias in the sequence composition of the dataset. in the rdp, the non-coverage rates for all primers except 27f were ≪6%, while in the metagenomic datasets, most were ≫10%. if one considers that a single mismatch near the 3′ end of the primer might greatly reduce pcr efficiency, then some phylum non-coverage rates would change by more than 20%. primer binding-site sequence variants that could not pair with their corresponding primers are discussed.  our study revealed the potential bias introduced by the use of universal bacterial primers in the assessment of microbial communities. with the development of high-throughput, next-generation sequencing techniques, it will become feasible to sequence more of the hypervariable regions of the bacterial 16s rrna gene. this, in turn, will lead to the more frequent use of the primers discussed here. 