Accepted Article
Evaluation of revised PCR primers for more inclusive quantification of ammonia-oxidizing archaea and bacteria1
Kelley A. Meinhardta, Anthony Bertagnollia, Manmeet Pannua, Stuart Stranda, Sally Brownb, David A. Stahla#
Civil and Environmental Engineeringa, School of Environmental and Forest Sciencesb, University of Washington, Seattle, Washington, USA
Running Head: Revised primers for AOA and AOB quantification
#Address correspondence to David A. Stahl, [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1758-2229.12259
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Summary
Ammonia-oxidizing archaea (AOA) and bacteria (AOB) fill key roles in the nitrogen cycle. Thus, well-vetted methods for characterizing their distribution are essential for framing studies of their significance in natural and managed systems. Quantification of the gene coding for one subunit of the ammonia monooxygenase (amoA) by PCR is frequently employed to enumerate
the two groups. However, variable amplification of sequence variants comprising this conserved genetic marker for ammonia oxidizers potentially compromises within- and between-system comparisons. We compared the performance of newly designed non-degenerate qPCR primer sets to existing primer sets commonly used to quantify the amoA of AOA and AOB using a collection of plasmids and soil DNA samples. The new AOA primer set provided improved quantification of model mixtures of different amoA sequence variants and increased detection of
amoA in DNA recovered from soils. Although both primer sets for the AOB provided similar results for many comparisons, the new primers demonstrated increased detection in environmental application. Thus, the new primer sets should provide a useful complement to primers now commonly used to characterize the environmental distribution of AOA and AOB.
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Introduction
In general, ammonia-oxidizing archaea (AOA) greatly outnumber their bacterial counterparts (AOB) in diverse habitats, including marine and freshwater systems (Francis et al., 2005; Herrmann et al., 2008), wetlands (Höfferle et al., 2010), hot springs (De La Torre et al., 2008;
Hatzenpichler et al., 2008), and various soil types (Leininger et al., 2006; He et al., 2007; Kemnitz et al., 2007; Boyle-Yarwood et al., 2008; Nicol et al., 2008). While nitrification inhibition studies (Offre et al., 2009; Lehtovirta-Morley et al., 2013) and the cultivation of a few
AOA (Konneke et al., 2005; Martens-Habbena et al., 2009; Lehtovirta-Morley et al., 2011; Tourna et al., 2011) have informed their environmental significance, the relative contribution of
this newly-discovered assemblage to nitrification in different environments in relation to AOB remains an open area of investigation.
Of particular relevance to this question is the significance of AOA and AOB in agricultural systems. Agricultural lands currently cover 38% of the Earth’s terrestrial surface (Foley et al., 2011), and anthropogenic inputs of reactive N (primarily as NH4+ and NO3-) to these lands will increase with rising food demands. Anticipated investments in bioenergy crop cultivation will also increase the need for reactive N in agronomic systems. Reliance on added synthetic N in managed soils has many environmental costs (McLauchlan, 2006; Snyder et al., 2009), including nitrate leaching, reduced soil fertility, and significant greenhouse gas emissions (Snyder et al., 2009; Brown et al., 2010). Mitigating the environmental impacts of intensive agriculture should
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therefore benefit from a better understanding of the functional significance of AOA and AOB and the abiotic and biotic factors controlling their distribution and activity.
Accurate enumeration of the two assemblages, as is commonly done by quantitative PCR (qPCR) of the amoA gene (Rotthauwe et al., 1997; Francis et al., 2005; Leininger et al., 2006; Di et al., 2009; Kelly et al., 2011; Petersen et al., 2012), is required to identify controlling
environmental variables. Apart from the known concerns of variable DNA extraction efficiency (Leininger et al., 2006; Smith et al., 2006), co-extracted polymerase inhibitors, and variable gene copy number, commonly used primer sets were designed using a much more limited reference collection of archaeal and bacterial amoA sequences than is now available, and thus may not uniformly capture the diversity of these two major populations (Jia and Conrad, 2009; GubryRangin et al., 2010; Pester et al., 2012).
A common design feature of PCR primers targeting a coding sequence that is diagnostic for a functional assemblage, such as the amoA, is the incorporation of degenerate positions to improve coverage of sequence variation in the primer binding sequence. However, this design strategy is known to contribute to differential amplification of sequence variants (Polz and Cavanaugh, 1998). In addition, the use of highly degenerate primers generally requires a lower hybridization stringency during the primer annealing step (Linhart and Shamir, 2002), contributing to possible amplification of non-target sequences. Thus, the goal of this study was to design more generally-inclusive, non-degenerate primer sets for quantification of AOA and AOB in various environments and objectively compare their performance with commonly used primer sets. Primer design considered sequences from soil metagenomes (Table 1; Bertagnolli et al., in
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review), cultured representatives, and over 20 environmental clone libraries. Rather than introduce degenerate positions, uniform amplification and specificity was achieved by extending the primers to encompass a larger conserved target site and by selecting nucleotides anticipated to form less-destabilizing mismatches in positions of known nucleotide variability (Fig. S1). Efficiency and amplification specificity were evaluated using a collection of plasmids containing amoA gene sequences that encompassed the priming regions representative of major clades of soil AOA and cultured AOB. Comparisons to commonly used primers (Fig. 1), here designated as FranAOA (Francis et al., 2005), TreuAOA (Treusch et al., 2005), and MincAOA (Mincer et al., 2007) for AOA and RottAOB (Rotthauwe et al., 1997) for AOB, were conducted using individual plasmids, plasmid mixtures, and environmental DNA samples (see the Supporting Information for further details).
Results
Specificity of newly designed archaeal and bacterial amoA primer sets. The specificity of both new qPCR primer sets was confirmed via in silico and molecular analysis. See the Supporting Information for further details.
Reaction parameters and statistics. For AOA amoA quantification, a plasmid containing a 660 bp AOA amoA gene fragment with fewest mismatches to all primer sets (Fig. S2) was used as the standard. An equimolar mixture of two plasmids containing a 673 bp amoA sequence from Nitrosomonas europaea and Nitrosospira briensis was used for AOB quantification. The
standard curves were linear over five orders of magnitude (R2 = 0.993 to 0.999), with a detection
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limit of 100 copies per reaction. The average efficiency of all AOA quantification reactions with the FranAOA, TreuAOA, MincAOA, and GenAOA primer sets was 84%, 99%, 86, and 95%, respectively. Average reaction efficiencies were 90% for the RottAOB and 97% for the GenAOB primer set. See the Supporting Information for AOA and AOB reaction statistics for all primer sets and for confirmation of amplification specificity via melt curve and gel analysis.
Assessment of archaeal amoA quantification accuracy using single plasmid templates. There were significant differences in average archaeal amoA gene counts from the four archaeal
primer sets when quantifying 11 amoA sequence variants, relative to expected total plasmid counts determined using a primer set targeting the TOPO plasmid backbone (87% reaction efficiency, see Table 2 for primer sequences; χ2 = 17.48, P = 0.002; Fig. 2). While individual
statistical analyses were not conducted, the results suggested primer set amplification bias for some variants. For example, Plasmids 3, 4, and 9 (See Fig. 2 for clade designations of sequence variants) were not uniformly amplified by the four primer sets. The counts produced with the FranAOA and GenAOA primer sets were closest to the expected counts; however the FranAOA counts were generally higher and the GenAOA counts lower. The GenAOA primer set amplified seven of the 11 plasmids within the range determined with the TOPO primer set, with no instance of over-amplification (Fig. 2). The MincAOA primer set amplified two of the 11 within the TOPO count range, but all other counts from this and the other primer sets were either above or below the anticipated count. The average gene count range for the reactions with the GenAOA primer set was the lowest, with the ranges for the MincAOA, TreuAOA, and FranAOA primer sets being five-, 10-, and 15-fold larger.
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Archaeal amoA quantification in plasmid mixtures. Four simplified communities comprised of a mixture of two or more plasmids (from the study set of 11) were quantified to test the capability of each primer set to uniformly amplify multiple diverse variants. The average number of archaeal amoA genes amplified from the four plasmid mixtures was significantly
affected by the primer set (P < 0.0001), and there was a significant interaction effect of the primer set and the mixture on the average gene count (P < 0.0001; Fig. 3). With mixture 1, which contained the more- and less-efficiently amplified plasmids (determined using all primer
sets; 80:20), the average amoA gene count determined with the TreuAOA and GenAOA primer sets was comparable to the vector count, lower with the FranAOA primer set, and higher with the MincAOA primer set (Fig. 3a). The average gene counts obtained from mixture 2 (more- and less-efficiently amplified plasmid, 20:80) and mixture 3 (equimolar mixture of four soil clade
representatives), were lower than the vector count for all primer sets, with greater disparity observed with mixture 2 (Fig. 3b and c). Quantification of the mixture of all 11 plasmids (mixture 4) was similar for the four primer sets (Fig. 3d), but lower than the vector count. The gene count range determined from duplicate reactions was small for all primer sets except TreuAOA.
Archaeal amoA quantification in soil DNA samples. The archaeal amoA gene count in soil DNA samples varied with primer set (Fig. 4; P = 0.007). The TreuAOA and MincAOA primer sets tended to generate higher gene counts than the FranAOA and GenAOA primer sets (Fig. 4), most notably for the PR samples. Possible over-amplification by the TreuAOA primer set was observed with the OM samples, as the AOA amoA gene counts using this primer set were much higher than those produced using the other three primer sets.
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Bacterial amoA quantification using individual plasmids and plasmid mixtures. The primer set significantly affected the bacterial amoA gene count from individual plasmids and plasmid mixtures (Fig. S3; P < 0.0001), and there was also a significant primer by mixture interaction (P < 0.0001). Reactions containing 100% Nsp briensis plasmid amplified equally well using either the RottAOB or GenAOB primer set and were consistent with the vector counts. However, differential amplification by the two primer sets was observed when Nm europaea comprised 100% of the template or was included in a mixture (mixtures 1 and 2). This was primarily due to less efficient amplification of Nm europaea gene sequences by the GenAOB primer set and slight over-amplification by the RottAOB primer set relative to the vector count.
Bacterial amoA quantification in soil DNA samples. There was a significant primer set effect
(P < 0.0001) and a significant primer set-by-sample interaction (P < 0.0001) on the average
bacterial amoA gene counts amplified from the eight soil DNA samples (Fig. 5). The average
bacterial amoA gene counts from five of the eight samples were similar for the RottAOB and GenAOB primer sets. Gene counts varied with the other three samples (PAT1, OM1-2), with higher counts generated by the GenAOB primer set. This higher abundance may be due to a broader detection of AOB by the new primer set.
Amplification of archaeal and bacterial amoA from isolates and diverse environments. The GenAOA primer set successfully amplified amoA sequences from three marine archaeal isolates, Nitrosopumilus maritimus SCM1, HCA1, and PS0 (Qin et al., 2014), without non-target amplification, though strain HCA1 was not as strongly amplified as the other two isolates. When
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using DNA from an alpine soil, the GenAOA primer set amplified a specific product without non-target amplification. The GenAOB primer set also amplified the target in this soil, but in some samples, minor non-target amplification was observed in the melt curves and via gel electrophoresis (data not shown).
Archaeal and bacterial amoA clone library and metagenomic community comparisons. In general, archaeal amoA sequences obtained from soil DNA using the GenAOA primer set were
more similar to those in a metagenome than those amplified with the FranAOA primer set (Figure 6a). The abundance of Nitrosotalea subclade 1.1 members in the GenAOA clone library (82%) was more similar to that in the metagenome (58%), than those recovered using the FranAOA primer set (37%). Additionally, sequences affiliated with the Nitrososphaera 54d9 fosmid-containing soil clade were over-represented by the FranAOA primer set relative to the metagenome. The FranAOA primer set did, however, capture the Nitrososphaera 2.1 clade, also
found in the metagenome, but not detected by the GenAOA primer set. Neither primer set recovered sequences affiliated with Nitrosotalea subclade 2 present in the metagenome.
Bacterial community comparisons of the clone libraries and metagenome were limited by the low number of bacterial amoA reads found in the metagenome (6 reads) and poor cloning efficiency of RottAOB amplicons. Among the limited number of sequences recovered, the GenAOB primer set revealed a representative of the Nitrosospira 8A clade not detected by the RottAOB primer set. However, the RottAOB primer set captured members of Nitrosospira 1
clades not detected by the GenAOB primer set (Figure 6b). The RottAOB library was dominated
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by Nitrosospira 2 clade members, whereas the GenAOB library consisted largely of unclassified
Nitrosospira 2 and Nitrosospira 3A.2 and 2 clade members.
Discussion
Considering the much greater diversity of ammonia-oxidizing microorganisms revealed through ongoing environmental surveys, it was uncertain whether primers designed earlier using a more limited data set of amoA sequences provide appropriate coverage or are suitable for qPCR. Thus, we used metagenomic and clone library sequence data from soils collected across Washington, as well as amoA sequences from environmental and cultured AOA and AOB, to design a set of non-degenerate primers for each group. The utility of the new primer sets was vetted through testing a collection of cloned amoA sequence variants representative of cultured
and environmental sequence diversity and by comparative analyses of soil DNA.
Comparative amplification of a study set of 11 plasmid clones of archaeal amoA sequence variants, singly and in mixtures, showed greater variability in amplification efficiency to be associated with primer sets containing a high number of degeneracies. Similar but less comprehensive comparative analyses were used to characterize the AOB amoA primer sets. Since the GenAOB primer set was designed to provide general coverage of common soil AOB phylotypes, it was less efficient in amplifying the amoA from Nm europaea than the RottAOB primer set. However, the GenAOB was more selective than the RottAOB primer set, since the
latter tended toward over-amplification. The unexpected non-target amplification by the RottAOB primer set may reflect reduced stringency associated with two degenerate positions in
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this set of primers. Thus, the concerted use of both primer set may be advisable for developing a more complete census of diversity and abundance of AOB in different environmental and engineered systems.
Comparative quantitative analyses of AOA in soil DNA showed that gene count was affected by primer set, differing by up to one order of magnitude for certain primer sets, further emphasizing the importance of initial primer set evaluation for large-scale environmental studies. The poor performance of the TreuAOA primer set in studies of different plasmid mixtures was also reflected in the high variability and poor selectivity in environmental application. The gene counts yielded by both AOB primer sets were comparable for most samples, although, primer set significantly affected overall gene count. With some samples, the GenAOB primer set returned counts 2 x 105 to 6 x 106 higher than the RottAOB primer set. Thus, the RottAOB primer set may have been missing certain ammonia-oxidizing members of the soil communities studied. For example, with minor to moderate non-target amplification in some samples, the GenAOB primer set amplified amoA sequence from a Washington alpine soil not amplified by the
RottAOB primer set (data not shown). AOB abundance has been reported to be low or undetectable in alpine soils (Zhang et al., 2009; Alves et al., 2013). Thus, the high copy number returned by the new primer set in open environmental application, in combination with observed specific amplification, suggests that the GenAOB primer set provided better recovery of AOB populations relative to the RottAOB primer set when applied to soil communities. However, we note that the recovery of more sequence variants, alone, is not support for greater accuracy of general AOB detection, since the recovery of another variant may simply reflect biased amplification of a minor population member.
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The final assessment of primer set efficacy was based on a comparison of amoA sequence variants identified in a metagenome relative to amoA sequences recovered by cloning products amplified by the different primer sets. For the AOA, the metagenome diversity was better replicated by sequences amplified by the newly developed GenAOA primer set than by the FranAOA primer set. Although the new primers were designed using sequences largely from
soils, the inclusion of other AOA clade representatives and marine isolate sequences in primer design suggests that the new primer set will have utility in non-soil systems. Very few AOB sequences were recovered in the soil metagenome, limiting the conclusions that can be drawn about primer set performance. Although the RottAOB primer set detected more clades than the GenAOB primer set, given the sparse number of AOB amoA sequence types identified in the metagenome, it is unclear if this reflected better general coverage or selective amplification of less abundant phylotypes. Thus, further investigation is warranted to establish relative primer performance.
In summary, these comparative studies clearly demonstrated that AOA and AOB abundance data
is affected by primer set. Thus, variable and non-target amplification have almost certainly skewed the results of past studies based on commonly used primer sets. Although no single primer set can provide fully unbiased amplification of the different sequence variants present in an environmental or engineered system, we suggest that an initial comparative analysis of primer amplification will provide for improved cross-system comparisons. This is essential in order to correctly identify factors controlling the distribution and activity of ammonia-oxidizing populations in the environment.
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Experimental procedures
Soil DNA extraction, amoA amplification, clone library construction, and sequencing. After evaluating three extraction methods, DNA was extracted from 0.3 g homogenized soil using the MO BIO PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA). Two extractions were performed simultaneously and pooled. End-point PCR for AOA and AOB amoA amplification was carried out in 20 µL reactions following published thermoprofiles (Rotthauwe et al., 1997; Francis et al., 2005), and products were verified via agarose gel
electrophoresis. The amplicons were cloned and sequenced on an ABI 3730xl DNA Analyzer (ABI Life Sciences, San Diego, CA) from the M13F priming site.
Design of new archaeal and bacterial amoA primers. Primer design was based on published amoA sequences and sequences recovered from surface soil samples collected from five sites in Washington, USA, each with differing physical and chemical properties (Table 1). Clone library sequences (and sequences from each major clade in the ARB database for AOA) were used for internal design of GenAOAF and GenAOBR (Fig. 1; see Table 2 for all primer sequences). Following, environmental archaeal and bacterial amoA sequences within and flanking the Francis
Arch-amoAR and Rotthauwe amoA-1F target regions, respectively, were selectively recovered from soil DNA metagenomic sequence libraries (Bertagnolli et al., in review; Table 1) to design AOAextR and AOBextF. Extended -length amplicons or sequences from cultured
representatives were then used to design GenAOAR and amoA-1Fmod (Fig. 1).
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Comparative amplification of divergent archaeal and bacterial amoA sequences using single and mixed plasmid templates. The AOA and AOB amoA gene counts were obtained for
individual and mixed plasmids using the FranAOA, TreuAOA, MincAOA, and GenAOA primer set for AOA, or the RottAOB and GenAOB primer sets for AOB, and compared to a count determined using a TOPO vector-specific primer set. Reactions were performed in a Roche LightCycler Carousel-Based System with LightCycler FastStart DNA Master SYBR Green I (Roche, Indianapolis, IN) in glass capillaries. Melt curves and gel electrophoresis confirmed product specificity.
Comparative archaeal and bacterial amoA primer amplification of environmental DNA
samples. Each primer set was used to comparatively quantify total AOA and AOB amoA gene abundance in different soil samples. Marine isolates were quantified with the GenAOA primer set to demonstrate the applicability of this primer set in different environment.
Comparison of metagenomic data with communities captured by new and conventional primer sets. Clone libraries were constructed using the newly designed archaeal and bacterial primer sets for one DNA sample for which metagenomic and amplicon clone sequence data (FranAOB and RottAOB primer set amplification) was available (Bertagnolli et al., in review; Table 1). The resulting data allowed for comparison of the community captured by each primer set relative to the less-biased metagenomic community composition assessment.
Detailed experimental procedures can be found in the Supporting Information.
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Acknowledgements
This research was supported by the Genomic Science and Technology for Energy and the Environment grant DE-SC0006869 from the Department of Energy. We thank W. Qin for helpful discussions and B. Meyer and two anonymous reviewers for comments on an earlier version of this manuscript.
References
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Table 1. Soil sampling locations and descriptions. Samples
Location
Coordinates
Land use
Dominant vegetation
Soil typea
pHb
C:Nb
PR1-3
Prosser, WA
46.2556 N, 119.7326 W
Agriculture
Blackwell switchgrass (Panicum virgatum) Alfalfa (Medicago sativa)
Silt loam
8.2
9.6
PAT1-2
Paterson, WA
45.9412 N, 119.4867 W
Agriculture
Kanlow switchgrass
Sand
5.2
14.4
OM1
Lee Forest, WA
47.8208 N, 122.1233 W
Forest
Red alder (Alnus rubra) Black cottonwood (Populus trichocarpa)
Gravelly sandy loam
4.6
21.9
OM2
Everett, WA
47.9678 N, 122.2249 W
Forest
Black cottonwood
Silt loam
4.9
13.4
OM3
Pack Forest, WA
46.8434 N, 122.3170 W
Research Forest
Red alder Douglas Fir (Pseudotsuga menziesii)
Gravelly silt loam
6.0
15.6
a
Soil types provided by the United States Department of Agriculture, Natural Resources Conservation Service Web Soil Survey.
b
Average of replicate measurements from throughout the site.
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Table 2. Primers used in this study.
Primer set
Sequence (5’ – 3’)
FranAOA Arch-amoAF Arch-amoAR
STA ATG GTC TGG CTT AGA CG GCG GCC ATC CAT CTG TAT GT
TreuAOA amo196F amo277R
GGW GTK CCR GGR ACW GCM AC CRA TGA AGT CRT AHG GRT ADC C
MincAOA CrenAmoAQ-F CrenAmoAModR
GCA RGT MGG WAA RTT CTA YAA AAG CGG CCA TCC ATC TGT A
RottAOB amoA-1F amoA-2R
GGG GTT TCT ACT GGT GGT CCC CTC KGS AAA GCC TTC TTC
GenAOA GenAOAF GenAOAR
ATA GAG CCT CAA GTA GGA AAG TTC TA CCA AGC GGC CAT CCA GCT GTA TGT CC
GenAOB amoA-1Fmod GenAOBR
CTG GGG TTT CTA CTG GTG GTC GCA GTG ATC ATC CAG TTG CG
TOPO4 backbone TOPO4-2259F TOPO4-2448R
CCA TAG TTG CCT GAC TCC CC ATA GAC TGG ATG GAG GCG GA
AOAextR
GGG GTT TTA ATC CCA CTT
this study
AOBextF
GGG CGA CTG GGA TTT CTG G
this study
Reference (Francis et al., 2005)
(Treusch et al., 2005)
(Mincer et al., 2007)
(Rotthauwe et al., 1997)
this study
this study
this study
27 This article is protected by copyright. All rights reserved.
Evaluation of revised PCR primers for more inclusive quantification of ammonia-oxidizing archaea and bacteria1
Kelley A. Meinhardta, Anthony Bertagnollia, Manmeet Pannua, Stuart Stranda, Sally Brownb, David A. Stahla#
Civil and Environmental Engineeringa, School of Environmental and Forest Sciencesb, University of Washington, Seattle, Washington, USA
Running Head: Revised primers for AOA and AOB quantification
#Address correspondence to David A. Stahl, [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1758-2229.12259
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Summary
Ammonia-oxidizing archaea (AOA) and bacteria (AOB) fill key roles in the nitrogen cycle. Thus, well-vetted methods for characterizing their distribution are essential for framing studies of their significance in natural and managed systems. Quantification of the gene coding for one subunit of the ammonia monooxygenase (amoA) by PCR is frequently employed to enumerate
the two groups. However, variable amplification of sequence variants comprising this conserved genetic marker for ammonia oxidizers potentially compromises within- and between-system comparisons. We compared the performance of newly designed non-degenerate qPCR primer sets to existing primer sets commonly used to quantify the amoA of AOA and AOB using a collection of plasmids and soil DNA samples. The new AOA primer set provided improved quantification of model mixtures of different amoA sequence variants and increased detection of
amoA in DNA recovered from soils. Although both primer sets for the AOB provided similar results for many comparisons, the new primers demonstrated increased detection in environmental application. Thus, the new primer sets should provide a useful complement to primers now commonly used to characterize the environmental distribution of AOA and AOB.
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Introduction
In general, ammonia-oxidizing archaea (AOA) greatly outnumber their bacterial counterparts (AOB) in diverse habitats, including marine and freshwater systems (Francis et al., 2005; Herrmann et al., 2008), wetlands (Höfferle et al., 2010), hot springs (De La Torre et al., 2008;
Hatzenpichler et al., 2008), and various soil types (Leininger et al., 2006; He et al., 2007; Kemnitz et al., 2007; Boyle-Yarwood et al., 2008; Nicol et al., 2008). While nitrification inhibition studies (Offre et al., 2009; Lehtovirta-Morley et al., 2013) and the cultivation of a few
AOA (Konneke et al., 2005; Martens-Habbena et al., 2009; Lehtovirta-Morley et al., 2011; Tourna et al., 2011) have informed their environmental significance, the relative contribution of
this newly-discovered assemblage to nitrification in different environments in relation to AOB remains an open area of investigation.
Of particular relevance to this question is the significance of AOA and AOB in agricultural systems. Agricultural lands currently cover 38% of the Earth’s terrestrial surface (Foley et al., 2011), and anthropogenic inputs of reactive N (primarily as NH4+ and NO3-) to these lands will increase with rising food demands. Anticipated investments in bioenergy crop cultivation will also increase the need for reactive N in agronomic systems. Reliance on added synthetic N in managed soils has many environmental costs (McLauchlan, 2006; Snyder et al., 2009), including nitrate leaching, reduced soil fertility, and significant greenhouse gas emissions (Snyder et al., 2009; Brown et al., 2010). Mitigating the environmental impacts of intensive agriculture should
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therefore benefit from a better understanding of the functional significance of AOA and AOB and the abiotic and biotic factors controlling their distribution and activity.
Accurate enumeration of the two assemblages, as is commonly done by quantitative PCR (qPCR) of the amoA gene (Rotthauwe et al., 1997; Francis et al., 2005; Leininger et al., 2006; Di et al., 2009; Kelly et al., 2011; Petersen et al., 2012), is required to identify controlling
environmental variables. Apart from the known concerns of variable DNA extraction efficiency (Leininger et al., 2006; Smith et al., 2006), co-extracted polymerase inhibitors, and variable gene copy number, commonly used primer sets were designed using a much more limited reference collection of archaeal and bacterial amoA sequences than is now available, and thus may not uniformly capture the diversity of these two major populations (Jia and Conrad, 2009; GubryRangin et al., 2010; Pester et al., 2012).
A common design feature of PCR primers targeting a coding sequence that is diagnostic for a functional assemblage, such as the amoA, is the incorporation of degenerate positions to improve coverage of sequence variation in the primer binding sequence. However, this design strategy is known to contribute to differential amplification of sequence variants (Polz and Cavanaugh, 1998). In addition, the use of highly degenerate primers generally requires a lower hybridization stringency during the primer annealing step (Linhart and Shamir, 2002), contributing to possible amplification of non-target sequences. Thus, the goal of this study was to design more generally-inclusive, non-degenerate primer sets for quantification of AOA and AOB in various environments and objectively compare their performance with commonly used primer sets. Primer design considered sequences from soil metagenomes (Table 1; Bertagnolli et al., in
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review), cultured representatives, and over 20 environmental clone libraries. Rather than introduce degenerate positions, uniform amplification and specificity was achieved by extending the primers to encompass a larger conserved target site and by selecting nucleotides anticipated to form less-destabilizing mismatches in positions of known nucleotide variability (Fig. S1). Efficiency and amplification specificity were evaluated using a collection of plasmids containing amoA gene sequences that encompassed the priming regions representative of major clades of soil AOA and cultured AOB. Comparisons to commonly used primers (Fig. 1), here designated as FranAOA (Francis et al., 2005), TreuAOA (Treusch et al., 2005), and MincAOA (Mincer et al., 2007) for AOA and RottAOB (Rotthauwe et al., 1997) for AOB, were conducted using individual plasmids, plasmid mixtures, and environmental DNA samples (see the Supporting Information for further details).
Results
Specificity of newly designed archaeal and bacterial amoA primer sets. The specificity of both new qPCR primer sets was confirmed via in silico and molecular analysis. See the Supporting Information for further details.
Reaction parameters and statistics. For AOA amoA quantification, a plasmid containing a 660 bp AOA amoA gene fragment with fewest mismatches to all primer sets (Fig. S2) was used as the standard. An equimolar mixture of two plasmids containing a 673 bp amoA sequence from Nitrosomonas europaea and Nitrosospira briensis was used for AOB quantification. The
standard curves were linear over five orders of magnitude (R2 = 0.993 to 0.999), with a detection
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limit of 100 copies per reaction. The average efficiency of all AOA quantification reactions with the FranAOA, TreuAOA, MincAOA, and GenAOA primer sets was 84%, 99%, 86, and 95%, respectively. Average reaction efficiencies were 90% for the RottAOB and 97% for the GenAOB primer set. See the Supporting Information for AOA and AOB reaction statistics for all primer sets and for confirmation of amplification specificity via melt curve and gel analysis.
Assessment of archaeal amoA quantification accuracy using single plasmid templates. There were significant differences in average archaeal amoA gene counts from the four archaeal
primer sets when quantifying 11 amoA sequence variants, relative to expected total plasmid counts determined using a primer set targeting the TOPO plasmid backbone (87% reaction efficiency, see Table 2 for primer sequences; χ2 = 17.48, P = 0.002; Fig. 2). While individual
statistical analyses were not conducted, the results suggested primer set amplification bias for some variants. For example, Plasmids 3, 4, and 9 (See Fig. 2 for clade designations of sequence variants) were not uniformly amplified by the four primer sets. The counts produced with the FranAOA and GenAOA primer sets were closest to the expected counts; however the FranAOA counts were generally higher and the GenAOA counts lower. The GenAOA primer set amplified seven of the 11 plasmids within the range determined with the TOPO primer set, with no instance of over-amplification (Fig. 2). The MincAOA primer set amplified two of the 11 within the TOPO count range, but all other counts from this and the other primer sets were either above or below the anticipated count. The average gene count range for the reactions with the GenAOA primer set was the lowest, with the ranges for the MincAOA, TreuAOA, and FranAOA primer sets being five-, 10-, and 15-fold larger.
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Archaeal amoA quantification in plasmid mixtures. Four simplified communities comprised of a mixture of two or more plasmids (from the study set of 11) were quantified to test the capability of each primer set to uniformly amplify multiple diverse variants. The average number of archaeal amoA genes amplified from the four plasmid mixtures was significantly
affected by the primer set (P < 0.0001), and there was a significant interaction effect of the primer set and the mixture on the average gene count (P < 0.0001; Fig. 3). With mixture 1, which contained the more- and less-efficiently amplified plasmids (determined using all primer
sets; 80:20), the average amoA gene count determined with the TreuAOA and GenAOA primer sets was comparable to the vector count, lower with the FranAOA primer set, and higher with the MincAOA primer set (Fig. 3a). The average gene counts obtained from mixture 2 (more- and less-efficiently amplified plasmid, 20:80) and mixture 3 (equimolar mixture of four soil clade
representatives), were lower than the vector count for all primer sets, with greater disparity observed with mixture 2 (Fig. 3b and c). Quantification of the mixture of all 11 plasmids (mixture 4) was similar for the four primer sets (Fig. 3d), but lower than the vector count. The gene count range determined from duplicate reactions was small for all primer sets except TreuAOA.
Archaeal amoA quantification in soil DNA samples. The archaeal amoA gene count in soil DNA samples varied with primer set (Fig. 4; P = 0.007). The TreuAOA and MincAOA primer sets tended to generate higher gene counts than the FranAOA and GenAOA primer sets (Fig. 4), most notably for the PR samples. Possible over-amplification by the TreuAOA primer set was observed with the OM samples, as the AOA amoA gene counts using this primer set were much higher than those produced using the other three primer sets.
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Bacterial amoA quantification using individual plasmids and plasmid mixtures. The primer set significantly affected the bacterial amoA gene count from individual plasmids and plasmid mixtures (Fig. S3; P < 0.0001), and there was also a significant primer by mixture interaction (P < 0.0001). Reactions containing 100% Nsp briensis plasmid amplified equally well using either the RottAOB or GenAOB primer set and were consistent with the vector counts. However, differential amplification by the two primer sets was observed when Nm europaea comprised 100% of the template or was included in a mixture (mixtures 1 and 2). This was primarily due to less efficient amplification of Nm europaea gene sequences by the GenAOB primer set and slight over-amplification by the RottAOB primer set relative to the vector count.
Bacterial amoA quantification in soil DNA samples. There was a significant primer set effect
(P < 0.0001) and a significant primer set-by-sample interaction (P < 0.0001) on the average
bacterial amoA gene counts amplified from the eight soil DNA samples (Fig. 5). The average
bacterial amoA gene counts from five of the eight samples were similar for the RottAOB and GenAOB primer sets. Gene counts varied with the other three samples (PAT1, OM1-2), with higher counts generated by the GenAOB primer set. This higher abundance may be due to a broader detection of AOB by the new primer set.
Amplification of archaeal and bacterial amoA from isolates and diverse environments. The GenAOA primer set successfully amplified amoA sequences from three marine archaeal isolates, Nitrosopumilus maritimus SCM1, HCA1, and PS0 (Qin et al., 2014), without non-target amplification, though strain HCA1 was not as strongly amplified as the other two isolates. When
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using DNA from an alpine soil, the GenAOA primer set amplified a specific product without non-target amplification. The GenAOB primer set also amplified the target in this soil, but in some samples, minor non-target amplification was observed in the melt curves and via gel electrophoresis (data not shown).
Archaeal and bacterial amoA clone library and metagenomic community comparisons. In general, archaeal amoA sequences obtained from soil DNA using the GenAOA primer set were
more similar to those in a metagenome than those amplified with the FranAOA primer set (Figure 6a). The abundance of Nitrosotalea subclade 1.1 members in the GenAOA clone library (82%) was more similar to that in the metagenome (58%), than those recovered using the FranAOA primer set (37%). Additionally, sequences affiliated with the Nitrososphaera 54d9 fosmid-containing soil clade were over-represented by the FranAOA primer set relative to the metagenome. The FranAOA primer set did, however, capture the Nitrososphaera 2.1 clade, also
found in the metagenome, but not detected by the GenAOA primer set. Neither primer set recovered sequences affiliated with Nitrosotalea subclade 2 present in the metagenome.
Bacterial community comparisons of the clone libraries and metagenome were limited by the low number of bacterial amoA reads found in the metagenome (6 reads) and poor cloning efficiency of RottAOB amplicons. Among the limited number of sequences recovered, the GenAOB primer set revealed a representative of the Nitrosospira 8A clade not detected by the RottAOB primer set. However, the RottAOB primer set captured members of Nitrosospira 1
clades not detected by the GenAOB primer set (Figure 6b). The RottAOB library was dominated
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by Nitrosospira 2 clade members, whereas the GenAOB library consisted largely of unclassified
Nitrosospira 2 and Nitrosospira 3A.2 and 2 clade members.
Discussion
Considering the much greater diversity of ammonia-oxidizing microorganisms revealed through ongoing environmental surveys, it was uncertain whether primers designed earlier using a more limited data set of amoA sequences provide appropriate coverage or are suitable for qPCR. Thus, we used metagenomic and clone library sequence data from soils collected across Washington, as well as amoA sequences from environmental and cultured AOA and AOB, to design a set of non-degenerate primers for each group. The utility of the new primer sets was vetted through testing a collection of cloned amoA sequence variants representative of cultured
and environmental sequence diversity and by comparative analyses of soil DNA.
Comparative amplification of a study set of 11 plasmid clones of archaeal amoA sequence variants, singly and in mixtures, showed greater variability in amplification efficiency to be associated with primer sets containing a high number of degeneracies. Similar but less comprehensive comparative analyses were used to characterize the AOB amoA primer sets. Since the GenAOB primer set was designed to provide general coverage of common soil AOB phylotypes, it was less efficient in amplifying the amoA from Nm europaea than the RottAOB primer set. However, the GenAOB was more selective than the RottAOB primer set, since the
latter tended toward over-amplification. The unexpected non-target amplification by the RottAOB primer set may reflect reduced stringency associated with two degenerate positions in
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this set of primers. Thus, the concerted use of both primer set may be advisable for developing a more complete census of diversity and abundance of AOB in different environmental and engineered systems.
Comparative quantitative analyses of AOA in soil DNA showed that gene count was affected by primer set, differing by up to one order of magnitude for certain primer sets, further emphasizing the importance of initial primer set evaluation for large-scale environmental studies. The poor performance of the TreuAOA primer set in studies of different plasmid mixtures was also reflected in the high variability and poor selectivity in environmental application. The gene counts yielded by both AOB primer sets were comparable for most samples, although, primer set significantly affected overall gene count. With some samples, the GenAOB primer set returned counts 2 x 105 to 6 x 106 higher than the RottAOB primer set. Thus, the RottAOB primer set may have been missing certain ammonia-oxidizing members of the soil communities studied. For example, with minor to moderate non-target amplification in some samples, the GenAOB primer set amplified amoA sequence from a Washington alpine soil not amplified by the
RottAOB primer set (data not shown). AOB abundance has been reported to be low or undetectable in alpine soils (Zhang et al., 2009; Alves et al., 2013). Thus, the high copy number returned by the new primer set in open environmental application, in combination with observed specific amplification, suggests that the GenAOB primer set provided better recovery of AOB populations relative to the RottAOB primer set when applied to soil communities. However, we note that the recovery of more sequence variants, alone, is not support for greater accuracy of general AOB detection, since the recovery of another variant may simply reflect biased amplification of a minor population member.
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The final assessment of primer set efficacy was based on a comparison of amoA sequence variants identified in a metagenome relative to amoA sequences recovered by cloning products amplified by the different primer sets. For the AOA, the metagenome diversity was better replicated by sequences amplified by the newly developed GenAOA primer set than by the FranAOA primer set. Although the new primers were designed using sequences largely from
soils, the inclusion of other AOA clade representatives and marine isolate sequences in primer design suggests that the new primer set will have utility in non-soil systems. Very few AOB sequences were recovered in the soil metagenome, limiting the conclusions that can be drawn about primer set performance. Although the RottAOB primer set detected more clades than the GenAOB primer set, given the sparse number of AOB amoA sequence types identified in the metagenome, it is unclear if this reflected better general coverage or selective amplification of less abundant phylotypes. Thus, further investigation is warranted to establish relative primer performance.
In summary, these comparative studies clearly demonstrated that AOA and AOB abundance data
is affected by primer set. Thus, variable and non-target amplification have almost certainly skewed the results of past studies based on commonly used primer sets. Although no single primer set can provide fully unbiased amplification of the different sequence variants present in an environmental or engineered system, we suggest that an initial comparative analysis of primer amplification will provide for improved cross-system comparisons. This is essential in order to correctly identify factors controlling the distribution and activity of ammonia-oxidizing populations in the environment.
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Experimental procedures
Soil DNA extraction, amoA amplification, clone library construction, and sequencing. After evaluating three extraction methods, DNA was extracted from 0.3 g homogenized soil using the MO BIO PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA). Two extractions were performed simultaneously and pooled. End-point PCR for AOA and AOB amoA amplification was carried out in 20 µL reactions following published thermoprofiles (Rotthauwe et al., 1997; Francis et al., 2005), and products were verified via agarose gel
electrophoresis. The amplicons were cloned and sequenced on an ABI 3730xl DNA Analyzer (ABI Life Sciences, San Diego, CA) from the M13F priming site.
Design of new archaeal and bacterial amoA primers. Primer design was based on published amoA sequences and sequences recovered from surface soil samples collected from five sites in Washington, USA, each with differing physical and chemical properties (Table 1). Clone library sequences (and sequences from each major clade in the ARB database for AOA) were used for internal design of GenAOAF and GenAOBR (Fig. 1; see Table 2 for all primer sequences). Following, environmental archaeal and bacterial amoA sequences within and flanking the Francis
Arch-amoAR and Rotthauwe amoA-1F target regions, respectively, were selectively recovered from soil DNA metagenomic sequence libraries (Bertagnolli et al., in review; Table 1) to design AOAextR and AOBextF. Extended -length amplicons or sequences from cultured
representatives were then used to design GenAOAR and amoA-1Fmod (Fig. 1).
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Comparative amplification of divergent archaeal and bacterial amoA sequences using single and mixed plasmid templates. The AOA and AOB amoA gene counts were obtained for
individual and mixed plasmids using the FranAOA, TreuAOA, MincAOA, and GenAOA primer set for AOA, or the RottAOB and GenAOB primer sets for AOB, and compared to a count determined using a TOPO vector-specific primer set. Reactions were performed in a Roche LightCycler Carousel-Based System with LightCycler FastStart DNA Master SYBR Green I (Roche, Indianapolis, IN) in glass capillaries. Melt curves and gel electrophoresis confirmed product specificity.
Comparative archaeal and bacterial amoA primer amplification of environmental DNA
samples. Each primer set was used to comparatively quantify total AOA and AOB amoA gene abundance in different soil samples. Marine isolates were quantified with the GenAOA primer set to demonstrate the applicability of this primer set in different environment.
Comparison of metagenomic data with communities captured by new and conventional primer sets. Clone libraries were constructed using the newly designed archaeal and bacterial primer sets for one DNA sample for which metagenomic and amplicon clone sequence data (FranAOB and RottAOB primer set amplification) was available (Bertagnolli et al., in review; Table 1). The resulting data allowed for comparison of the community captured by each primer set relative to the less-biased metagenomic community composition assessment.
Detailed experimental procedures can be found in the Supporting Information.
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Acknowledgements
This research was supported by the Genomic Science and Technology for Energy and the Environment grant DE-SC0006869 from the Department of Energy. We thank W. Qin for helpful discussions and B. Meyer and two anonymous reviewers for comments on an earlier version of this manuscript.
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Qin, W., Amin, S.A., Martens-Habbena, W., Walker, C.B., Urakawa, H., Devol, A.H. et al. (2014) Marine ammonia-oxidizing archaeal isolates display obligate mixotrophy and wide ecotypic variation. Proceedings of the National Academy of Sciences 111: 12504-12509. Rotthauwe, J.H., Witzel, K.P., and Liesack, W. (1997) The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Applied and Environmental Microbiology 63: 4704-4712. Smith, C.J., Nedwell, D.B., Dong, L.F., and Osborn, A.M. (2006) Evaluation of quantitative polymerase chain reaction-based approaches for determining gene copy and gene transcript numbers in environmental samples. Environmental Microbiology 8: 804-815. Snyder, C.S., Bruulsema, T.W., Jensen, T.L., and Fixen, P.E. (2009) Review of greenhouse gas emissions from crop production systems and fertilizer management effects. Agriculture, Ecosystems & Environment 133: 247-266.
Tourna, M., Stieglmeier, M., Spang, A., Könneke, M., Schintlmeister, A., Urich, T. et al. (2011) Nitrososphaera viennensis, an ammonia oxidizing archaeon from soil. Proceedings of the National Academy of Sciences 108: 8420-8425.
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Treusch, A.H., Leininger, S., Kletzin, A., Schuster, S.C., Klenk, H.-P., and Schleper, C. (2005) Novel genes for nitrite reductase and Amo-related proteins indicate a role of uncultivated mesophilic crenarchaeota in nitrogen cycling. Environmental Microbiology 7: 1985-1995. Zhang, L.-M., Wang, M., Prosser, J.I., Zheng, Y.-M., and He, J.-Z. (2009) Altitude ammoniaoxidizing bacteria and archaea in soils of Mount Everest. FEMS Microbiology Ecology 70: 208217.
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EMI4_12259_F1
EMI4_12259_F2
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EMI4_12259_F3
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EMI4_12259_F4
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EMI4_12259_F5
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EMI4_12259_F6
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Table 1. Soil sampling locations and descriptions. Samples
Location
Coordinates
Land use
Dominant vegetation
Soil typea
pHb
C:Nb
PR1-3
Prosser, WA
46.2556 N, 119.7326 W
Agriculture
Blackwell switchgrass (Panicum virgatum) Alfalfa (Medicago sativa)
Silt loam
8.2
9.6
PAT1-2
Paterson, WA
45.9412 N, 119.4867 W
Agriculture
Kanlow switchgrass
Sand
5.2
14.4
OM1
Lee Forest, WA
47.8208 N, 122.1233 W
Forest
Red alder (Alnus rubra) Black cottonwood (Populus trichocarpa)
Gravelly sandy loam
4.6
21.9
OM2
Everett, WA
47.9678 N, 122.2249 W
Forest
Black cottonwood
Silt loam
4.9
13.4
OM3
Pack Forest, WA
46.8434 N, 122.3170 W
Research Forest
Red alder Douglas Fir (Pseudotsuga menziesii)
Gravelly silt loam
6.0
15.6
a
Soil types provided by the United States Department of Agriculture, Natural Resources Conservation Service Web Soil Survey.
b
Average of replicate measurements from throughout the site.
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Table 2. Primers used in this study.
Primer set
Sequence (5’ – 3’)
FranAOA Arch-amoAF Arch-amoAR
STA ATG GTC TGG CTT AGA CG GCG GCC ATC CAT CTG TAT GT
TreuAOA amo196F amo277R
GGW GTK CCR GGR ACW GCM AC CRA TGA AGT CRT AHG GRT ADC C
MincAOA CrenAmoAQ-F CrenAmoAModR
GCA RGT MGG WAA RTT CTA YAA AAG CGG CCA TCC ATC TGT A
RottAOB amoA-1F amoA-2R
GGG GTT TCT ACT GGT GGT CCC CTC KGS AAA GCC TTC TTC
GenAOA GenAOAF GenAOAR
ATA GAG CCT CAA GTA GGA AAG TTC TA CCA AGC GGC CAT CCA GCT GTA TGT CC
GenAOB amoA-1Fmod GenAOBR
CTG GGG TTT CTA CTG GTG GTC GCA GTG ATC ATC CAG TTG CG
TOPO4 backbone TOPO4-2259F TOPO4-2448R
CCA TAG TTG CCT GAC TCC CC ATA GAC TGG ATG GAG GCG GA
AOAextR
GGG GTT TTA ATC CCA CTT
this study
AOBextF
GGG CGA CTG GGA TTT CTG G
this study
Reference (Francis et al., 2005)
(Treusch et al., 2005)
(Mincer et al., 2007)
(Rotthauwe et al., 1997)
this study
this study
this study
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