Improved group-specific primers based on the full SILVA 16S rRNA
gene reference database
Supporting Information (SI)
Table S1
Test Prime analysis of published primer pairs and PCR exclusivity testing using strains
from target and non-target taxa as template
Table S2
Base pair positions with high target-taxon specificity
Table S3
Phylogenetic affiliation at the family level of 16S rRNA gene sequences from
rhizosphere soil clone libraries
Table S4
Isolates used for optimization of newly designed primers and exclusivity testing
Table S5
Earlier evaluations of primer pairs though sequencing of clone libraries
Table S6
Comparison of 454 data generated with universal primers and group-specific clone
libraries
Table S7
Percentage of sequence identities for both primers inside the target taxon in SILVA
Test Prime analysis
Fig. S1
ARB analysis of published primers
Fig. S2
Example of phylum/class specific T-RFLP electropherograms from rhizosphere sample
P2Fl5 (Peru, Flowering stage, plant No. 5) analyzed via GeneMapper
Table S1. TestPrime analysis of published primer pairs and PCR exclusivity testing using strains from target and non-target taxa as template
PrecisionA
Target group
Alphaproteobacteria
Betaproteobacteria
Gammaproteobacteria
Primer pair
AT
SensitivityB
0MM
1MM
1MM*
0MM
1MM
1MM*
3´end
(1MM)
Gammaproteobacteria
Bacteroidetes
Actinobacteria
Firmicutes
X
X
X
X
-
-
-
X
X
58
98.5
89.3
89.6
66.9
84.9
79.6
94.7
682F/908R
59
94
81.9
94.2
73.6
91.9
90.7
98.8
Eub338f/Bet680
60
98.8
59.8
59.7
82.5
89.9
89.3
99.4
X
X
-
Beta359f/Beta682r
60
99.4
86.9
86.8
76.5
91.6
91
99.4
-
X
X
-
-
-
Gam395f/Gam871r
54
98.6
81.3
81.2
42
72
71.2
99.2
-
-
X
-
-
-
CFB555f/cfb967R
61
99.8
99.2
99.2
70.9
88
87
99
798cfbF/cfb967R
61
93.1
80.6
80.5
80.2
88.1
86.8
98.7
S-C-Act-235-a-S-20/518r
54
93.2
80.3
81.5
55.9
79
66.3
87.3
X
X
-
X
X
X
X
-
X
X
-
Act920F3/Act1200R
61
81.1
30.5
30.4
81.7
89.9
89.1
99.2
Firm350f/Firm814r
59
99.8
92.1
92.4
20.6
29.1
28.9
99.8
GCLGC354f/Firm814r
59
99.8
97.5
97.6
20.2
28.2
28
99.8
61 97.3 82.6
AT, optimized annealing temperature
95.7
71.7
90.3
83
92.7
X
X
-
X
-
X
-
X
X
X
X
-
X
X
X
Actinobacteria
928F-Firm/1040FirmR
Abbreviation:
Betaproteobacteria
Alf28f/Alf684r
Bacteroidetes
Firmicutes
DNA from bacterial isolates belonging to phylogenetic class/group
Alphaproteobacteria
…Percentage of sequence identities for both primers restricted to the target taxon in SILVA Test Prime analysis (SSU Ref 114 NR) using 3 different parameters
0MM…no mismatch allowed
1MM…1 mismatch per primer allowed
1MM*…1 mismatch per primer allowed, with a no mismatch zone at the last 3 bases from 3´ -end
B
…Percentage of sequence identities for both primers inside the target taxon in SILVA Test Prime analysis
3´end(1MM)…percentage of sequences with no mismatch at the 3 terminal bases from 3´ -end, when 1 mismatch is allowed
A
Legend
X
X
-
PCR product of correct size was amplified from a target strain
PCR product of correct size was amplified from a non-target strain
No PCR amplification
Table S2. Base pair positions with high target-taxon specificity
Primer
positionC
0
98,9
14/1226
0
97,6
29/1226
3
9
97,6
29/1226
ADF681f
2
ALF968
1
19
9
17
6
92,7
97,6
92,3
97,8
94/1226
29/1226
94/1226
27/1226
908R
0
-
-
-
S-C-bProt-0972-a-S-18
1
13
98,2
21/1181
S-C-bProt-1221-a-A-17
2
13
98,2
20/1181
14
93,2
76/1181
Firmicutes
Actinobacteria
Bacteroidetes
S-C-aProt-0528-a-S-19
S-C-aProt-0689-a-A-22
1
SpecificityD
Alphaproteobacteria
Target-specific
baseB
Betaproteobacteria
PrimerA
Beta359f
0
-
-
-
Beta682r
1
8
97
35/1181
S-P-Bdet-0107-a-S-21
2
1
20
99,4
91,4
7/1237
113/1237
S-P-Bdet-0309-a-A-21
2
0
96,5
43/1237
1
7
8
0
1
0
0
0
5
7
99,7
99
98,9
97,8
96,9
96,9
99,7
96,2
94,9
96,1
95,1
99,4
4/1237
12/1237
14/1237
27/1237
38/1237
38/1237
3/1168
44/1168
60/1168
46/1168
57/1168
7/1168
CFB555f
2
cfb967R
2
CFB986r
798cfbF
S-P-Acti-0927-a-S-17
1
0
1
S-P-Acti-1154-a-S-19
4
S-P-Acti-1339-a-A-18
1
Act920F3
0
-
-
-
Act1200R
-
S-C-Act-235-a-S-20
0
0
-
-
S-P-Firm-0352-a-S-18
2
0
99,8
3/1271
3
94,9
71/1271
S-P-Firm-0525-a-S-18
1
0
99,6
5/1271
928F-Firm
0
-
-
-
1040FirmR
0
-
-
-
Firm814r
0
0
-
-
-
-
-
-
0
-
-
-
Gam395f
Gam871r
A
Bases of published primers analyzed were used in exclusivity assays (Table 3, 4); Taxon-specific bases of primers 682F, Alf684r,
Bet680, Firm350f and GCLGC354f are covered by the analysis of ADF681f (682F , Alf684r), Beta682r (for Bet680) and SP-Firm 0352-a-S18 (GCLGC354f, Firm350f)
B
C
Number of bases with a precision > 90% in the RSet database used for primer design; N…0 bases
Position of specific (>90%) base in primer counting from the 3´ -end (3´ -end position = 0)
D
Left column: Primer specificity in percent values; Right column: false positives/non-target sequences in the representative database
Table S3. Phylogenetic affiliation at the family level of 16S rRNA gene sequences from rhizosphere soil clone
libraries
Target group
Family
No of clones
Total
Aurantimonadaceae
Bradyrhizobiaceae
Brucellaceae
Caulobacteraceae
Erythrobacteraceae
Hyphomicrobiaceae
Methylobacteriaceae
Phyllobacteriaceae
Rhizobiaceae
Rhodobacteraceae
Sphingomonadaceae
Xanthobacteraceae
Beijerinckiaceae
Methylocystaceae
Unclassified Alphaproteobacteria
Potato rhizosphere
Hyphomicrobiaceae
Caulobacteraceae
Aurantimonadaceae
Beijerinckiaceae
Bradyrhizobiaceae
Methylobacteriaceae
Phyllobacteriaceae
Rhizobiaceae
Rhodobacteraceae
Acetobacteraceae
Rhodospirillaceae
Erythrobacteraceae
Sphingomonadaceae
Brucellaceae
Potato rhizosphere
Total
Burkholderiaceae
Piscinibacter
Rubrivivax
Nitrosomonadaceae
Oxalobacteriaceae
Comamonadaceae
Leptothrix
Alcaligenaceae
Unclassified Betaproteobcateria
176
4
3
2
19
5
9
1
16
8
4
55
11
1
2
36
75
7
3
1
2
4
9
3
8
4
1
2
3
27
1
Primers
Alphaproteobacteria
S-C-aProt-0528-a-S-19
S-C-aProt-0689-a-A-22
Alphaproteobacteria
S-C-aProt-0528-a-S-19
ALF968
Betaproteobacteria
S-C-bProt-0972-a-S-18
S-C-bProt-1221-a-A-17
91
21
1
2
1
25
24
5
1
10
Betaproteobacteria
Beta359f
S-C-bProt-1221-a-A-17
Actinobacteria
S-P-Acti-1154-a-S-19
S-P-Acti-1339-a-A-18
Actinobacteria
S-P-Acti-0927-a-S-17
S-P-Acti-1339-a-A-18
Bacteroidetes
Human feces
Sutterellaceae
Potato rhizosphere
Comamonadaceae
Alcaligenaceae
Oxalobacteraceae
Rhodocyclaceae
Burkholdericeae
Unclassified Betaproteobacteria
Potato rhizosphere
Thermomonosporaceae
Micromonosporaceae
Pseudonocardiaceae
Micrococcaceae
Geodermatophilaceae
Catenulisporaceae
Cellulomonadaceae
Promicromonosporaceae
Actinosynnemataceae
Microbacteriaceae
Micromonosporaceae
Mycobacteriaceae
Nocardioidaceae
Intrasporangiaceae
Streptomycetaceae
Streptosporangiaceae
Human feces
Bifidobacteriaceae
Potato rhizosphere
Acidimicrobiaceae
Bogoriellaceae
Cellulomonadaceae
80
80
45
9
8
16
1
4
12
68
3
1
3
12
9
1
1
3
1
9
4
5
5
2
2
6
Geodermatophilaceae
Intrasporangiaceae
Microbacteriaceae
Micrococcaceae
Micromonosporaceae
Mycobacteriaceae
Nocardiaceae
Nocardioidaceae
Pseudonocardiaceae
Streptomycetaceae
Thermomonosporaceae
Unclassified Actinobacteria
Potato rhizosphere
Flavobacteriaceae
Cytophagaceae
12
1
7
4
3
1
2
12
7
1
1
18
173
94
21
61
76
1
1
5
S-P-Bdet-0107-a-S-21
S-P-Bdet-0309-a-A-21
Bacteroidetes
S-P-Bdet-0107-a-S-21
cfb967R
Firmicutes
S-P-Firm-0352-a-S-18
S-P-Firm-0525-a-A-18
Firmicutes
S-P-Firm-0525-a-S-18
1040FirmR
Sphingobacteriaceae
Cyclobacteriaceae
Unclassified Bacteroidetes
Human feces
Bacteroidaceae
Porphorymonadaceae
Prevotellaceae
Rikenellaceae
Potato rhizosphere
Cytophagaceae
Flavobacteriaceae
Sphingobacteriaceae
unclassified Bacteroidetes
Potato rhizosphere
Bacillaceae
Paenibacillaceae
Bacillus
Geobacillus
Human feces
Veillonellaceae
Streptococcaceae
Ruminococcaceae
Lactobacillaceae
Lachnospiraceae
Enterococcaceae
Clostridiaceae
Acidaminococcaceae
Potato rhizosphere
Bacillaceae
Planococcaceae
Clostridiaceae
Geobacillus
Bacillus
23
1
34
51
11
10
4
26
59
10
33
1
15
54
52
2
49
3
51
23
10
1
2
2
1
9
3
85
78
5
1
1
77
Table S4. Isolates used for optimization of newly designed primers and exclusivity testing
Genus of closest Blastn affiliation of isolate
(based on 16S rRNA gene)
Alphaproteobacteria
Betaproteobacteria
Gammaproteobacteria
Bacteroidetes
Actinobacteria
Firmicutes
Sphingomonas
Sphingomonas
Acetobacter
Agrobacterium
Burkholderia
Comamonas
Variovorax
Pseudomonas
Pseudomonas
Stenotrophomonas
Margalefia
Enterobacter
Flavobacterium
Chryseobacterium
Microbacterium
Oerskovia
Leifsonia
Curtobacterium
Arthrobacter
Bacillus subtilis
Paenibacillus
Bacillus simplex
Accession nr. of
closest Blastn
hit
EF153191
AM998824
FM178867
FJ830830
GQ478268
HM113649
JF683273
EU196391
AB602399
EF204217
AY702662
AB435591
FN645724
AM232811
DQ365561
AB562466
AB278552
EU236753
AF197055
DQ122328
EU496554
EF690433
Table S5 Earlier evaluations of primer pairs though sequencing of clone libraries
Percentage of
target-specificity
100
100
Number of
clones
20
50
Environment
Reference
mesotrophic coastal sea
oligotrophic ocean
(Mühling et al., 2008)
682F/908R
100
20
marine biofilms
Eub338f/Bet680
Beta359f/Beta682r
96
100
30
100
100
100
100
20-25
20
50
20
50
20
50
soil
mesotrophic coastal sea
oligotrophic ocean
mesotrophic coastal sea
oligotrophic ocean
mesotrophic coastal sea
oligotrophic ocean
798cfbF/cfb967R
97
37
marine biofilms
S-C-Act-235-a-S-20/518r
60
20-25
soil
Act920F3/Act1200R
83
6
marine biofilms
Firm350f/Firm814r
20-25
mesotrophic coastal sea
oligotrophic ocean
GCLGC354f/Firm814r
70
0
-
928F-Firm/1040FirmR
56
35
Primer pair
Alf28f/Alf684r
Gam395f/Gam871r
CFB555f/CFB968r
(Bacchetti De Gregoris
et al., 2011)
(Fierer et al., 2005)
(Mühling et al., 2008)
(Mühling et al., 2008)
(Mühling et al., 2008)
(Mühling et al., 2008)
(Bacchetti De Gregoris
et al., 2011)
(Fierer et al., 2005)
(Bacchetti De Gregoris
et al., 2011)
(Mühling et al., 2008)
(Mühling et al., 2008)
marine biofilms
(Bacchetti De Gregoris
et al., 2011)
Table S6. Comparison of 454 data generated by the universal bacterial primers 8f/518 and group-specific clone
libraries
Bacteroidetes
Rhizosphere (2000 reads)
8f/518r
S-P-Bdet-0107-a-S-21
S-P-Bdet-0309-a-A-21
S-P-Bdet-0107-a-S-21
cfb967R
Number of different sequences summarized at the family level
Porphyromonadaceae
Flavobacteriaceae
Cyclobacteriaceae
Cytophagaceae
Saprospiraceae
Sphingobacteriaceae
Human feces samples (2000 reads)
Bacteroidaceae
Porphyromonadaceae
Prevotellaceae
Rikenellaceae
2
41
6
4
2
5
8f/518r
NA
75
1
18
NA
18
NA
25
NA
9
NA
1
S-P-Bdet-0107-a-S-21
S-P-Bdet-0309-a-A-21
+
+
NA
NA
+
+
+
+
8f/518r
S-C-aProt-0528-a-S-19
S-C-aProt-0689-a-A-21
Alphaproteobacteria
Rhizosphere Peru (2000 reads)
S-C-aProt-0528-a-S-19
ALF968
Number of different sequences summarized at the family level
Caulobacteraceae
Aurantimonadaceae
Beijerinckiaceae
Bradyrhizobiaceae
Brucellaceae
Hyphomicrobiaceae
Methylobacteriaceae
Phyllobacteriaceae
Rhizobiaceae
Xanthobacteraceae
Rhodobacteraceae
Acetobacteraceae
Rhodospirillaceae
Erythrobacteraceae
Sphingomonadaceae
Methylocystaceae
Human feces samples (2000 reads)
No families detected with both methods
14
NA
1
9
NA
7
6
5
4
NA
18
5
6
2
16
NA
15
4
1
3
1
6
1
15
7
2
3
NA
NA
5
45
2
8f/518r
S-C-aProt-0528-a-S-19
S-C-aProt-0689-a-A-21
NA
NA
2
1
2
4
1
5
5
3
8
NA
3
1
2
3
22
NA
Firmicutes
454 data
8f/518r
Rhizosphere Peru (2000
reads)
12
1
4
1
21
3
40
3
1
5
1
Human feces samples (2000 reads)
Clostridiaceae
Enterococcaceae
Erysipelotrichaceae
Lachnospiraceae
Lactobacillaceae
Ruminococcaceae
Streptococcaceae
Veillonellaceae
Families detected in
454 reads
Family present in 454 reads was detected in clone
library
+
+
+
+
+
+
+
+
454 data (Peru)
8f/518r
Rhizosphere Peru/Ecuador
Families detected in
454 data
Actinosynnemataceae
Geodermatophilaceae
Borgoriellaceae
Cellulomonadaceae
Catenulisporaceae
Intrasporangiaceae
Kineosporaceae
Microbacteriaceae
Micrococcaceae
Micromonosporaceae
Mycobacteriaceae
Nakamurellaceae
Nocardiaceae
Nocardioidaceae
Promicromonosporaceae
Propionibacteriaceae
Pseudonocardiaceae
Sanguibacteraceae
Streptomycetaceae
Thermomonosporaceae
Patulibacteraceae
S-P-Firm-0525-a-S-18
1040FirmR
Number of different sequences summarized at the family level
Bacillaceae
Paenibacillaceae
Planococcaceae
Staphylococcaceae
Clostridiaceae
Peptostreptococcaceae
Ruminococcaceae
Actinobacteria
Group-specific clone libraries
S-P-Firm-0352-a-S-18
S-P-Firm-0525-a-A-18
4
6
NA
NA
NA
3
NA
2
1
1
NA
1
1
2
10
1
NA
1
1
NA
1
+
+
NA
+
+
+
+
+
Group-specific primer pair (Ecuador)
S-P- S-P-Acti-01154-a-S-19
Acti-1339-a-A-18
S-P- S-P-Acti-0972-a-S-17
Acti-1339-a-A-18
Families detected in clone libraries
+
+
+
+
NA
+
+
+
+
+
+
NA
+
+
+
NA
+
NA
+
+
NA
+
+
NA
+
+
+
NA
+
+
+
+
NA
+
+
+
+
+
+
+
+
NA
Rubrobacteraceae
1
Acidothermaceae
NA
Human feces samples (2000 reads)
No families detected
NA
1
NA
NA
Group-specific primer pair
Betaproteobacteria
454 data (Peru)
8f/518r
Rhizosphere Peru / Ecuador
Rhodocyclaceae
Burkholderiaceae
Oxalobacteraceae
Comamonadaceae
Human feces samples
(2000 reads)
Sutturellaceae
Abbreviation/Legend:
Families detected in 454
data
S-C-bProt-0972-a-S-18
S-C-bProt-1221-a-A-17
Beta359f
S-C-bProt-1221-a-A-17
Families detected in clone libraries
1
+
+
14
+
+
6
+
+
10
+
+
8f/518r
+
NA, family was not detected
+, family was detected
S-C-bProt-0972-a-S-18
S-C-bProt-1221-a-A-17
+
Table S7. Percentage of sequence identities for both primers inside the target taxon in SILVA Test Prime analysis
TestPrime 8f /518r
Group
Alphaproteobacteria
Betaproteobacteria
Bacteroidetes
Actinobacteria
Firmicutes
0MM
1MM
1MM*
3´-end (1MM)
58.1
60.5
65.8
54.2
68.7
84.3
85.7
88
82.6
90
78.7
83
85.8
70.2
82.1
94.4
97.3
97.8
87.6
92.1
0MM…no mismatch allowed
1MM…1 mismatch per primer allowed
1MM*…1 mismatch per primer allowed, with a no mismatch zone at the last 3 bases from 3´ -end
3´end(1MM)…percentage of sequences with no mismatch at the 3 terminal bases from 3´ -end, when 1 mismatch is allowed
Figure S1.
Fig. S1.
ARB analysis of published primers. Primers are matched against Sequence Alignment
Information (SAI) calculated from all representatives of target and non-target phylum/class in the SILVA
SSU Ref 102 NR database. Each base pair position is evaluated: Dots (.) indicate a rate of >95 %
matches in the target group; small letters >70% < 95%; x < 70%; IUPAC codes are used when more than
one base occurs in > 30% of representatives; White letters in black boxes indicates mismatches towards
the primer
IUPAC ambiguity codes: R (A/G), Y (C/T), M (A/C), K (T/G), W (T/A), S (C/G), B (C/T/G), D (A/T/G), H
(A/T/C), V (A/C/G), N (A/C/G/T)
Fig. S2. A-E. Example of phylum/class specific T-RFLP electropherograms from one potato rhizosphere,
sampled at the plant flowering stage in Peru, analyzed via GeneMapper.
Abbreviation: FAM, 6-carboxyfluorescin-labeled primer