1
Supporting Information
2
3
Introduction
4
5
There are three primer sets frequently employed for AOA amoA quantification: Arch-amoAF
6
and Arch-amoAR (called the FranAOA primer set), were designed in 2005 using a limited set of
7
archaeal sequences and not originally intended for application in qPCR. The second set,
8
amo196F and amo277R, was designed by Treusch and others also in 2005 using archaeal amoA
9
sequences from soils (called the TreuAOA primer set). The third primer set, CrenAmoAQ-F and
10
CrenAmoAModR, published by Mincer and colleagues in 2007 was based on marine archaeal
11
sequences (called the MincAOA primer set). While the two later sets were designed specifically
12
for use in qPCR and produce small amplicons, they include numerous degenerate positions.
13
14
The most commonly used primer set to quantify AOB is amoA-1F and amoA-2R, developed by
15
Rotthauwe and others in 1997 (called the RottAOB primer set). These primers were not
16
specifically designed for qPCR, and the reverse primer contains two central degenerate positions.
17
18
Results
19
20
Specificity of newly designed archaeal and bacterial amoA primer sets. The specificity of
21
both new qPCR primer sets was confirmed via in silico and molecular analysis. A BLASTN
22
search of the NCBI website (NCBI) against the GenAOAF primer returned matches of 96-100%
23
identity (E value 9.0E-5) only to the ammonia monooxygenase (amoA) gene of thaumarchaeotes
1
24
and crenarchaeotes. The GenAOAR primer matched with 96-100% identity (E value 0.021) to
25
only the amoA of archaea and thaumarchaeotes. The amoA-1Fmod primer (for AOB) matched
26
with 100% identity (E value 0.028) only to the amoA of uncultured bacteria or to ammonia-
27
oxidizing beta proteobacteria, and the GenAOBF primer matched with 100% identity (E value
28
0.11) only to the amoA of uncultured bacteria. All amplicons were verified for correct size on
29
agarose gels and by sequencing of cloned amplification products.
30
31
Reaction parameters and statistics. A plasmid containing the 660 bp AOA amoA gene
32
fragment with fewest mismatches to all primer sets was used as the standard for AOA amoA
33
quantification. The standard curve produced a linear response over five orders of magnitude (R2
34
= 0.993 to 0.999, range for all reactions with all primer sets) with a detection limit of 10-100
35
copies per reaction. Loss of linearity below this was likely due to the formation of primer dimers
36
with some primer sets (often TreuAOA, occasionally MincAOA). For consistency, 100 copies
37
per reaction was selected as the lowest concentration in the standard series. The average
38
efficiency of all AOA quantification reactions with the FranAOA, TreuAOA, MincAOA, and
39
GenAOA primer sets was 84%, 99%, 86, and 95%, respectively. The lowest efficiency observed
40
(76%) was with the MincAOA primer set when quantifying the plasmid mixes, and the highest
41
observed (107%) was with the TreuAOA primer set when quantifying the single plasmids.
42
43
An equimolar mixture of two plasmids, each containing the 673 bp AOB amoA amplicon from
44
Nitrosomonas europaea and Nitrosospira briensis, was used as a standard and to determine the
45
reaction efficiency of the bacterial amoA primer sets. These two plasmids were mixed in order
46
to introduce a reasonable amount of sequence diversity within the standard (Nm europaea has
2
47
five mismatches between the two priming sites versus one mismatch in each of the Nsp briensis
48
priming sites). This plasmid mixture resulted in a linear amplification response (R2 = 0.994-
49
0.999 range for all reactions), with both the RottAOB and GenAOB primer sets having a
50
detection limit of 100 copies per reaction. Average reaction efficiencies were 90% for the
51
RottAOB and 97% for the GenAOB primer set. Efficiencies as low as 77% have been reported
52
when quantifying AOB in soils (Schauss et al., 2009), but values commonly range from 80% to
53
slightly over 100% (Nicol et al., 2008; Di et al., 2009; Jia and Conrad, 2009; Schauss et al.,
54
2009).
55
56
Assessment of archaeal amoA amplification specificity via melt curve and gel analysis. The
57
specificity of each primer set in all qPCR assays was first assessed by observing product melt
58
curves. For the single plasmids, the product melt curves for the MincAOA, TreuAOA, and
59
GenAOA primer sets were narrow and unimodal. However, those for the FranAOA primer set
60
were bi-modal. For the mixed plasmid products, the melt curves for the MincAOA and
61
GenAOA primer sets were narrow and unimodal, but the TreuAOA products were bimodal and
62
multimodal for the FranAOA products. Melt curves from soil DNA samples were unimodal only
63
when the MincAOA primer set was used and for all samples but one (OM3) when the GenAOA
64
primer set was used. Conversely, the product melt curves from the TreuAOA and FranAOA
65
primer sets were multimodal for all samples and often had low amplitude. Melt curves for
66
standards were always sharp and unimodal, suggesting no problems with the standards or
67
reaction conditions.
68
3
69
The specificity of each primer set was next evaluated by visualization of the qPCR products on
70
an agarose gel (Fig. S4). This was conducted to reveal any potential misinterpretations of primer
71
specificity based solely on melt curves. In general, each primer set preferentially amplified one
72
product of expected size, however, depending on the primer set and the samples being analyzed
73
(plasmids or soil DNA), non-target amplification did occur, as was suggested by earlier
74
observation of the product melt curves. When amplifying AOA amoA genes from plasmids
75
(single or mixed), the FranAOA primer set consistently co-amplified a ~1 kb product. With soil
76
DNA samples, the FranAOA primer set produced an intense target band but also amplified many
77
small (< 500 bp) non-target fragments, especially from the OM samples. Visualization of the
78
TreuAOA primer set amplicons from plasmids showed single, discrete bands for all samples,
79
with some primer dimer present in the non-template control (NTC) and lower standard reactions.
80
With the soil DNA samples, a single product amplified by the TreuAOA primer set was observed
81
for samples PR1-3 and PAT1-2, but little to no target product was apparent in OM samples.
82
Instead, larger non-target fragments were observed. The MincAOA primer set chiefly amplified
83
the target fragment from plasmids, but three other, non-target bands were occasionally present.
84
With the soil DNA samples, the MincAOA primer set amplified many non-target fragments,
85
occasionally more efficiently than the target. Strangely, this observation was inconsistent with
86
the results of the melt curve analysis, which provided little indication of non-target amplification.
87
Lastly, the GenAOA primer set produced a single product from both single and mixed plasmid
88
templates and most soil DNA samples. Product bands appeared thicker for a few soil DNA
89
samples, suggesting similar migration of nearly identical-sized amplicons.
90
4
91
Assessment of bacterial amoA amplification specificity via melt curve and gel analysis. The
92
AOB amoA plasmid and soil DNA qPCR products were evaluated for specificity by melt curve
93
analysis. With the plasmid mixes, the product melt curves from the RottAOB primer set were
94
bimodal for the mixed samples and unimodal for the samples containing 100% of either Nsp
95
briensis or Nm europaea plasmid. The melt curves of the standards, containing a 50:50 mixture
96
of the two plasmids, were also bimodal. Primer dimer was detected in the NTC for the RottAOB
97
primer set. The product melt curves from the GenAOB primer set were unimodal for all
98
plasmids, and no primer dimer was detected in the NTC. The melt curves from one soil DNA
99
sample indicated the presence of non-target amplification with the RottAOB primer set. The
100
product melt curves for the GenAOB primer were unimodal, indicating no non-target
101
amplification. Again, primer dimer was detected with the RottAOB primer set in the NTC but
102
not with the GenAOB primer set.
103
104
Gel visualization of the qPCR products from the plasmids and soil DNA samples confirmed the
105
characteristics suggested by the melt curves for the AOB amoA amplicons. The plasmid qPCR
106
products from the RottAOB primer set showed a dominance of the target amplicon, but a ~1 kb
107
amplicon was co-amplified (Fig. S5). The TOPO sequence was verified to contain no target for
108
the RottAOB primer sequences, so this non-target amplification is definitely unexpected. This
109
larger product was not apparent in the melt curve analysis. The GenAOB primer set produced a
110
single, correct-sized product for all plasmid samples. Imaging of the RottAOB qPCR product
111
from the soil DNA samples verified the presence of non-target amplicons in one sample, OM3
112
(Fig. S6). A single amplicon was observed in all soil DNA sample products amplified using the
113
GenAOB primer set.
5
114
115
Experimental procedures
116
117
Soil DNA extraction, amoA amplification, clone library construction, and sequencing.
118
Primer design was based on published amoA sequences and sequences recovered from eight
119
surface (0-15 cm) soil samples collected from five sites in Washington, USA, each with differing
120
physical and chemical properties (Table 1). After evaluating three DNA extraction methods by
121
comparing total DNA yield, extent of DNA shearing, and amplification inhibition over a range of
122
dilutions (data not shown), DNA was extracted from 0.3 g homogenized soil using the MO BIO
123
PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA) with the bead-beating
124
step being completed in a FastPrep-24 Instrument (MP Biomedicals, Irvine, CA). The bead
125
beating speed and time (5.5 m/s for 30 s) was optimized by assessing total DNA yield and the
126
extent of DNA shearing. Soil DNA was diluted below the concentration required for qPCR, and
127
no inhibition was detected in end-point PCR (data not shown). Two extractions were performed
128
simultaneously and pooled.
129
130
End-point PCR was carried out in 20 µL reactions with final concentrations of 1X GoTaq Green
131
Master Mix (Promega Inc., Madison, WI), 0.4 ng/µL Bovine Serum Albumin, 0.25 µM of each
132
primer, and 10-20 ng sample DNA. Reactions were run in a BioRad C1000 thermal cycler
133
(BioRad, Hercules, CA). The FranAOA primer set and published thermoprofile (Francis et al.,
134
2005) provided the longest amplicon of all the available primers sets and were used to amplify
135
the AOA amoA in each sample. The RottAOB primer set and published thermoprofile
136
(Rotthauwe et al., 1997) were used to amplify the AOB amoA in each sample. Resulting
6
137
amplicons (635 and 491 bp, respectively) were verified via agarose gel electrophoresis. The
138
PCR product was cleaned using the QIAquick PCR Purification kit (Qiagen, Valencia, CA),
139
cloned using the TOPO TA Cloning Kit with the pCR4-TOPO TA vector (Life Technologies,
140
Carlsbad, CA), and transformed into One Shot TOP10 Chemically Competent E. coli cells (Life
141
Technologies, Carlsbad, CA). After blue-white screening, 96 positive clones were picked into
142
100 µL of selective LB broth with 10% glycerol (final conc.) in culture plates. Plasmid
143
isolations and Sanger sequencing from the M13F priming site were performed on an ABI 3730xl
144
DNA Analyzer (ABI Life Sciences, San Diego, CA) by HTSeq, Inc. (Seattle, WA).
145
146
Design of archaeal amoA forward and bacterial amoA reverse primers. A total of 938 and
147
724 amoA clone library sequences for AOA and AOB, respectively, were used for internal
148
primer design. Three to five representative sequences from each major clade in the ARB
149
database were also selected for AOA primer design. The sequence variation in Nitrosopumilus
150
maritimus SCM1 and three new marine isolates was also considered. Separate alignments of
151
AOA and AOB amoA sequences were generated using the ClustalW Alignment option in the
152
Geneious 6.1.5 software package (Biomatters Ltd.) and by manual inspection. A conserved
153
region 85 nucleotides interior to the Arch-amoAR priming site, and in the same region targeted
154
by the Mincer CrenAmoAQ-F primer (Fig. 1), was selected for the design of a new archaeal
155
amoA forward qPCR primer, GenAOAF (see Table 2 for all primer sequences). Likewise for
156
AOB, GenAOBR was designed in a conserved region 80 nucleotides interior to the beginning of
157
the amoA-1F forward priming site (Fig. 1).
158
7
159
Selective recovery of environmental archaeal amoA sequences spanning the Arch-amoAR
160
primer binding site. Environmental archaeal amoA sequences within and flanking the Arch-
161
amoAR (FranAOA) target region were selectively recovered from soil DNA metagenomic
162
sequence libraries (Bertagnolli et al., in review; Table 1). Briefly, approximately 16 Gbp of
163
metagenomic data were generated from the eight soil DNA samples using the Illumina HiSeq
164
2000 platform. amoA reads were identified in cleaned metagenomic sequence data through
165
alignment (BLASTN, BLAST+ version 2.28) against AOA and AOB databases (Altschul et al.,
166
1990). The primer AOAextR was designed after retrieval and alignment of over 190
167
metagenomic reads mapping near the Arch-amoAR priming site.
168
169
A clone library was constructed using the Arch-amoAF and AOAextR primers to amplify a 660
170
bp amoA fragment from selected soil DNA samples, as described above (50 °C annealing
171
temperature). In consideration of 85 resulting clone sequences presenting natural variation
172
within the Arch-amoAR target region in these field sites, and preliminary testing with several
173
primer variants (data not shown), we designed a new primer, GenAOAR (Table 2), by modifying
174
Arch-amoAR and extending its binding region (Figure 1).
175
176
Comparative amplification of divergent archaeal amoA sequences using single and mixed
177
plasmid templates. Two experiments were conducted using a collection of cloned amoA
178
sequence variants to evaluate the specificity and relative stoichiometry of amplification using the
179
newly designed GenAOAF and GenAOAR primers (hereafter called the GenAOA primer set)
180
and the other commonly used primer sets. A study set of 11 clones of the 660 bp amplicons
181
representative of environmental sequence variation within the GenAOAF and GenAOAR
8
182
priming sites were used for comparative analyses. In the first experiment, the AOA amoA gene
183
count obtained from the amplification of individual plasmids using the FranAOA, TreuAOA,
184
MincAOA, and GenAOA primer set was compared to the count determined using TOPO vector-
185
specific primer set. A TOPO primer set (TOPO4-2259F and TOPO4-2448R; Table 2) was
186
designed to provide an objective gene copy number unaffected by insert sequence diversity.
187
Plasmids were diluted to a concentration of 0.25 ng/µL, and a total of 1 ng (4 µL) of plasmid
188
DNA was added to a 6 µL reaction mixture containing 1X LightCycler FastStart DNA Master
189
SYBR Green I (Roche, Indianapolis, IN), 4.5 mM MgCl2 (optimized), and 0.5 µM of each
190
primer (final concentrations) in LightCycler glass capillaries. Reactions were performed in a
191
Roche LightCycler Carousel-Based System with software v3.5. The qPCR thermoprofile for the
192
GenAOA primer set was 95 C for 5 min; 45 cycles of 95 C for 10 s, 55 C (optimized by
193
gradient end-point PCR with multiple environmental samples and plasmids) for 10 s, 72 C for
194
13 s, followed by a single 3 s data collection at 84 C, increased to avoid detection of possible
195
primer-dimer fluorescence. Melt curves (65 °C held for 15 s with continuous measurement to
196
95°C), as well as product electrophoresis on an agarose gel, confirmed product specificity.
197
Thermoprofiles for the TreuAOA and MincAOA primer sets were as published (Treusch et al.,
198
2005; Mincer et al., 2007), respectively, and the thermoprofile for the FranAOA primer set
199
followed that published by Di and colleagues (Di et al., 2009). Optimization of these
200
thermoprofiles was attempted after initial reaction efficiencies were lower than reported by the
201
respective authors, but for the soils tested in this study, conditions could not be improved. For
202
all primer sets, the initial 95 °C enzyme activation time was adjusted to 5 minutes, optimized for
203
the Taq used in these experiments.
204
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205
The second qPCR comparison experiment evaluated the relative efficiencies of each archaeal
206
amoA primer set to amplify four simplified model communities, comprised of a mixture of two
207
or more plasmids. Composition of mixtures was determined by natural sequence diversity and
208
the results of an initial examination of amplification efficiency. One mixture included two
209
plasmids in which the relative concentration of the most poorly amplifying sequence type was
210
present in a ratio of either 80:20 or 20:80 with a sequence type amplifying with good efficiency.
211
A second mixture consisted of equimolar amounts of plasmids representing four of the five
212
dominant soil clades (Nitrososphaera 54d9, Nitrososphaera subclade 1.1, unclassified
213
Nitrososphaera clade 1 (non-54d9), and Nitrosotalea subclade 1.1; Bertagnolli et al., in review)
214
observed in our sampling sites. A final mixture consisted of all 11 plasmids mixed at equimolar
215
ratios. Reactions were carried out in duplicate as described above.
216
217
Comparative archaeal amoA primer amplification of soil DNA samples. Each primer set
218
was also used to quantify the total AOA amoA gene abundance in environmental soil samples.
219
DNA from eight soil samples (Table 1) was amplified with each of the four primer sets, as
220
detailed above, to survey the overall gene count variation, error, and non-target amplification
221
among them. Melt curve analysis was conducted, and products were analyzed on agarose gels to
222
check for non-target amplification.
223
224
To reduce variability associated with enzyme age or reagent stocks (Smith et al., 2006), each
225
comparative analysis was conducted in one to two days with a single aliquot of MgCl2, each
226
primer, and enzyme and SYBR Green mixture. A plasmid containing the 660 bp AOA amoA
227
amplicon with the fewest mismatches in each priming site served as the internal standard (106-
10
228
102 copies, duplicate reaction for 102 copies). Accuracy was improved by including a standard
229
in every assay, as opposed to applying a single standard to multiple assays. Although the use of
230
external standards is often reported (Treusch et al., 2005; Park et al., 2008; Di et al., 2009), a
231
single standard does not account for inter-assay variation caused by the experimenter,
232
preparation, reagents, or the reaction itself (Smith et al., 2006). This plasmid returned the best
233
overall efficiency for all reactions with all primer sets in preliminary qPCR tests (data not
234
shown). All samples were run in duplicate.
235
236
Redesign of the bacterial amoA forward primer. We aimed to pair amoA-1F either
237
unchanged or a similar sequence, in which no degeneracies would be included, with GenAOBR
238
to capture the greatest sequence diversity present within an AOB community. The design of a
239
new primer flanking the amoA-1F primer hybridization site was based on natural sequence
240
variation within this priming site found in soil metagenomic sequences. Since the number of
241
suitable metagenomic sequences was low (11 in total), publically-available, complete bacterial
242
amoA sequences from Nitrosospira multiformis, Nsp briensis, Nitrosomonas europaea, Nm
243
eutropha, Nm sp. Is79A3, and Nm sp. AL212 (accession numbers NC007614.1, U76553.1,
244
NC_004757.1, NC_008344.1, NC_015731.1, NC_015222.1, respectively), were also included
245
for the design. The alignment was used to design a new forward primer (AOBextF, Table 2)
246
beginning 161 nucleotides upstream of the amoA-1F priming site (Fig. 1). This primer is similar
247
to (3 nucleotide differences), and hybridizes to the same region as, the A189F primer developed
248
by Holmes and colleagues (Holmes et al., 1995). The amoA-2R and newly designed AOBextF
249
primers were then used (as described above, 58° C annealing temperature) to amplify a 673 bp
250
fragment from two cultured AOB species, Nm europaea and Nsp briensis. On the basis of initial
11
251
amplification results (data not shown), amoA-1F was modified by three nucleotides to create
252
amoA-1Fmod (Table 2).
253
254
Comparative bacterial amoA amplification using plasmids and soil DNA samples. The two
255
plasmids containing the 673 bp amplicons of AOB amoA gene sequences from Nm europaea and
256
Nsp briensis, and a set of soil DNA samples, were used to compare the RottAOB primer set with
257
that of amoA-1Fmod and GenAOBR (hereafter called the GenAOB primer set). The two
258
plasmids were quantified separately and in mixtures (80:20 Nm europaea: Nsp briensis and vice
259
versa) with the RottAOB, GenAOB, and TOPO primer set, following the reaction preparation
260
methods described above for the AOA primer sets. Additionally, bacterial amoA genes were
261
quantified in the same eight soil DNA samples used to evaluate the archaeal amoA primer sets.
262
The thermoprofile used with the GenAOB primer set was identical to that used for the GenAOA
263
primer set but with annealing at 58 °C. The qPCR thermoprofile for the RottAOB primer set
264
followed the protocol of Di and colleagues (Di et al., 2009), with an initial Taq activation of 5
265
min at 95 °C. The plasmids were quantified with the TOPO primer set, as described above.
266
Samples were run in duplicate and the internal standard was prepared as above but using a 50:50
267
mixture of the Nm europaea and Nsp briensis 673 bp amoA amplicon insert plasmids.
268
269
Utility of newly designed archaeal and bacterial amoA primer sets for isolates and diverse
270
environments. The efficacy of the new primer sets to amplify archaeal and bacterial amoA
271
sequences from different environments was tested by end-point and quantitative PCR. DNA
272
from three marine archaeal isolates (Qin et al., in press) was used to assess the specificity of the
273
new archaeal primer set to marine AOA in end-point PCR, following the methods described
12
274
above. Additionally, DNA from alpine soils was used to verify amplification and specificity of
275
both new primer sets in another environment using the aforementioned qPCR methods.
276
277
Comparison of metagenomic data with communities captured by new and conventional
278
primer sets. Clone libraries were constructed using the newly designed archaeal and bacterial
279
primer sets for one of the DNA samples (PAT1; Table 1) for which metagenomic and amplicon
280
clone sequence data (FranAOA and RottAOB primer set amplification) was available
281
(Bertagnolli et al., in review; Table 1). The resulting data allowed for comparison of the
282
community captured by each primer set relative to the less-biased metagenomic community
283
composition assessment.
284
285
Statistical analyses. The data from qPCR assays of the plasmids and plasmid mixtures are
286
presented as copies per ng of plasmid DNA. Raw data from qPCR assays of soil DNA samples
287
were converted to cells per g soil. For AOB, cells numbers were estimated by dividing gene
288
counts by 2.6, accounting for 2 copies of the amoA gene in each genome and 1.3 genomes per
289
cell (Hermansson and Lindgren, 2001). When normally distributed, data were analyzed by one-
290
or two-way (when testing for an interaction) analysis of variance (ANOVA), followed by Tukey-
291
Kramer post hoc HSD comparisons to pinpoint significant differences among primer sets. The
292
AOA and AOB amoA soil DNA gene count data were log-transformed to normality. If the data
293
could not be successfully transformed to normality, the nonparametric Kruskal-Wallis test was
294
used. These analyses were performed in the statistical software package SPSS 19.0 with a
295
significance cut-off of P ≤ 0.05. Taxonomic assignment of AOA and AOB were based on the
13
296
work of Pester and others (2012) and Avrahami and Conrad (2003), respectively (Bertagnolli et
297
al., in review).
298
299
References
300
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Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. (1990) Basic Local
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Di, H.J., Cameron, K.C., Shen, J.P., Winefield, C.S., O/'Callaghan, M., Bowatte, S., and He, J.Z.
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(2009) Nitrification Driven by Bacteria and Not Archaea in Nitrogen-Rich Grassland Soils.
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16
359
360
Table S1. qPCR reaction statistics for AOA primer sets and experimental comparisons.
Primer set
Comparison
Slope
Efficiency (%)
Intercept
R2
FranAOA
Single plasmids
Plasmid mixes
Soil DNA samples
-3.63
-3.86
-3.80
88.5
81.5
83.3
39.48
39.51
38.70
-0.999
-0.998
-0.999
TreuAOA
Single plasmids
Plasmid mixes
Soil DNA samples
-3.17
-3.46
-3.41
107.0
94.7
96.3
39.48
39.54
39.40
-0.996
-0.995
-0.999
MincAOA
Single plasmids
Plasmid mixes
Soil DNA samples
-3.67
-4.07
-3.49
87.3
76.0
93.5
42.59
44.42
41.15
-0.993
-0.997
-0.995
GenAOA
Single plasmids
Plasmid mixes
Soil DNA samples
-3.55
-3.45
-3.34
91.2
94.9
99.3
40.03
39.44
38.72
-0.999
-0.999
-0.999
361
362
363
364
365
366
367
368
369
370
371
372
373
374
17
375
376
Table S2. qPCR reaction statistics for AOB primer sets and experimental comparisons.
Comparison
Slope
Efficiency (%)
RottAOB
Plasmid mixes
Soil DNA samples
-3.56
-3.61
91.0
89.3
38.27
37.46
-0.999
-0.999
GenAOB
Plasmid mixes
Soil DNA samples
-3.35
-3.43
98.8
95.6
38.82
39.08
-0.994
-0.999
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
18
Intercept
R2
Primer set
396
Supporting figure legends
397
398
Figure S1. An alignment of the GenAOA primer set (reverse complement of GenAOAR shown)
399
and 36 extended AOA amoA amplicons, representing the observed sequence diversity of a larger
400
set of sequences, shows variable positions within the priming region and intentionally-selected
401
nucleotide mismatches in the final primer design. For a) the forward primer, a variant including
402
a G nucleotide at the position indicated with a “1,” and for b) the reverse primer, three variants
403
including a T, G, or T at the position indicated with a “1,” “2,” or “3,” respectively, were
404
designed to attempt to further improve differential amplification of sequence variants with
405
multiple mismatches. Empirical testing via qPCR yielded inferior results (higher differential
406
amplification of sequence variants) compared with the final primer designs reported.
407
408
Figure S2. The extended-length (660 bp) AOA amoA amplicon used as a standard in qPCR
409
assays had the fewest number of mismatches for all primer sets. Mismatches within each primer
410
to the standard sequence are highlighted in color, while degeneracies are highlighted gray.
411
412
Figure S3. The average bacterial amoA gene count amplified from different ratios of Nsp
413
briensis and Nm europaea was significantly affected by the primer set. The GenAOB primer set
414
under-amplified Nm. europaea genes, while the RottAOB primer set slightly over-ampilfied
415
them, compared with the count produced using the TOPO primer set (range indicated by
416
horizontal gray bar). Data are means with range.
417
19
418
Figure S4. Archaeal amoA qPCR product generated from soil DNA using the a) GenAOA, b)
419
FranAOA, c) TreuAOA, and d) MincAOA primer sets visualized on a 2% agarose gel, 120 v for
420
45 min, stained with SYBR Safe DNA Gel Stain. In all gels, lane 1: 100 bp ladder, lane 2: 1 x
421
103 standard plasmid copies product, lanes 3-5: PR1-3 sample product, lanes 6-7: PAT1-2
422
sample product, lane 8: Boardman, WA soil DNA sample product (not discussed herein), lanes
423
9-11: OM1-3 sample product, lane 12: negative control, lane 10: 1,000 bp ladder.
424
425
Figure S5. Bacterial amoA qPCR product generated from single plasmids and plasmid mixtures
426
using the RottAOB and GenAOB primer sets visualized on a 2% agarose gel, 120 v for 45 min,
427
stained with SYBR Safe DNA Gel Stain. Lane 1 is 100 bp ladder, lanes 2-7 are product with the
428
RottAOB primer set, and lanes 9-14 are product with the GenAOB primer set. Products from
429
both primer sets follow the same order: 1 x 103 standard plasmid copies product, 100% Nsp
430
briensis, 100% Nm europaea, 20:80 Nm europaea:Nsp briensis, and 80:20 Nm europaea:Nsp
431
briensis (single and mixed plasmids that correspond to data in Figure S3), negative control.
432
433
Figure S6. Bacterial amoA qPCR product generated from soil DNA using the a) GenAOB and b)
434
RotthAOB primer sets visualized on a 2% agarose gel, 120 v for 45 min, stained with SYBR
435
Safe DNA Gel Stain. In both gels, lane 1: 100 bp ladder, lane 2: 1 x 103 standard plasmid copies
436
product, lanes 3-5: PR1-3 sample product, lanes 6-7: PAT1-2 sample product, lane 8: Boardman,
437
WA soil DNA sample product (not discussed herein), lanes 9-11: OM1-3 sample product, lane
438
12: negative control, lane 10: 1,000 bp ladder.
439
440
20
441
442
443
444
Figure S1.
445
446
447
448
449
450
451
452
21
453
454
455
Figure S2.
456
457
458
459
460
461
462
463
22
464
465
466
Figure S3.
467
468
469
470
471
472
473
474
475
476
477
478
479
480
23
481
482
483
484
Figure S4.
485
486
487
488
489
490
491
492
493
494
495
496
24
497
498
499
Figure S5.
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
25
516
517
518
Figure S6.
519
520
26