Supplementary Information: Endospore-enriched sequencing approach reveals unprecedented
diversity of Firmicutes in sediments
Tina Wunderlin, Thomas Junier, Ludovic Roussel-Delif, Nicole Jeanneret, Pilar Junier*
Laboratory of Microbiology, Institute of Biology, University of Neuchatel, CH-2000, Neuchâtel,
Switzerland
*
Corresponding author.
Mailing address: Laboratory of Microbiology, Institute of Biology,
University of Neuchâtel, CH - 2000 Neuchâtel
Phone: +4132 7182244
Fax: +4132 7182231
E-mail: pilar.junier@unine.ch
E-mail co-authors:
tina.wunderlin@unine.ch
thomas.junier@unine.ch
ludovic.roussel-delif@unine.ch
nicole.jeanneret@unine.ch
Supplementary Figure 1. Growth curves verifying the effectiveness of the disruption method
12 applied to different bacterial species. Growth curves of treated and control endospore
preparations from Bacillus subtilis, Bacillus megaterium, and Paenibacillus alvei, and a cell
culture of Escherichia coli. Treated = ○. Untreated = ●. Error bars from three independent
cultures. A combined treatment consisting of wet heat at 65°C for 20 min, followed by
lysozyme digestion for 60 min and digestion with 0.5 N NaOH and 1% SDS for another 60
min was applied to endospore preparations of B. subtilis, B. megaterium, and P. alvei and to
vegetative cells of E. coli. Growth of control cultures and re-growth of treated cultures was
measured as described above. Measurements were taken every 60 min during 15 hours.
Supplementary Figure 2. Schematic overview of the experimental procedure used to
determine diversity of endospore-forming bacteria in sediments. For the global bacterial
diversity the step of treatment to remove vegetative cells was omitted, to extract all bacteria in
the sediment.
Supplementary Figure 3. Principal component analysis (PCA) based on the community
structure of Firmicutes in the treated and untreated samples of Lake Geneva (LG) and Lake
Baikal (LB). Principal component analysis was done using R (Team" 2012) based on
normalized values of the community composition from Firmicutes.
Supplementary Table 1. Relative abundance of diversity on phylum level, based on 16S
rRNA sequencing. Values are given in percentage. Relative abundances of >1% are in bold.
Phylum
Firmicutes
Proteobacteria
Other
Chloroflexi
Acidobacteria
Actinobacteria
Planctomycetes
Nitrospirae
Gemmatimonadetes
Bacteroidetes
Cyanobacteria
AC1
Armatimonadetes
BRC1
Chlamydiae
Chlorobi
Elusimicrobia
FBP
Fibrobacteres
GN02
GN04
GOUTA4
Hyd24-12
LCP-89
Lentisphaerae
NC10
NKB19
OD1
OP1
OP11
OP3
OP8
OP9
SBR1093
SC4
Spirochaetes
TM6
TM7
TPD-58
Verrucomicrobia
WS1
WS2
WS3
WWE1
ZB3
Lake Geneva
whole
endosporecommunity
enriched
7.98
90.58
56.66
3.79
7.06
3.53
4.47
0.71
5.84
0.04
2.43
0.97
6.60
0.07
1.39
0.01
0.49
1.22
0.06
1.09
0.12
0.85
0.06
1.08
0.01
0.89
0.01
0.03
0.21
0.43
0.02
0.30
0.03
0.28
0.07
0.02
0.02
0.01
0.00
0.03
0.05
0.05
0.02
0.09
0.03
0.03
0.03
0.03
0.05
0.05
0.02
0.02
0.02
0.02
Lake Baikal
whole
endosporecommunity
enriched
19.04
83.92
42.42
5.94
10.59
5.94
5.60
0.64
4.69
0.35
4.39
2.09
2.80
0.38
1.89
0.17
1.49
0.19
0.76
0.02
0.66
0.11
0.53
0.05
0.35
0.03
0.43
0.05
1.06
0.03
0.58
0.03
0.33
0.63
0.02
0.15
0.35
0.03
0.08
0.18
0.15
0.13
0.15
0.10
0.13
0.05
0.03
0.02
0.08
0.01
0.02
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
Supplementary Table 2. Relative abundance (%) of diversity of Firmicutes determined by
16S rRNA sequencing. Values with relative abundance higher than 1 % are in bold.
Taxon
Firmicutes; Other
Bacilli; Other
Bacillales; Other
Alicyclobacillaceae; Other
Alicyclobacillus
Bacillaceae; Other
Anoxybacillus
Bacillus
Geobacillus
Marinibacillus
Oceanobacillus
Paenibacillaceae; Other
Cohnella
Paenibacillus
Planococcaceae; Other
Lysinibacillus
Paenisporosarcina
Rummeliibacillus
Solibacillus
Sporosarcina
Ureibacillus
Thermoactinomycetaceae;Other
Exiguobacteraceae
Trichococcus
Turicibacter
Clostridia; Other
Clostridiales; Other
Christensenellaceae
Clostridiaceae ;Other
02d06
Alkaliphilus
Caloramator
Clostridium
Oxobacter
Proteiniclasticum
SMB53
Sarcina
Dehalobacteriaceae; Dehalobacterium
Gracilibacteraceae; Other
Gracilibacter
Lachnospiraceae; Other
Lachnospiraceae; Clostridium
Coprococcus
Epulopiscium
Peptococcaceae; Desulfosporosinus
Pelotomaculum
Peptostreptococcaceae; Other
Peptostreptococcaceae; Clostridium
Tepidibacter
Peptostreptococcaceae; [Clostridium]
Ruminococcaceae; Other
Ruminococcaceae; Clostridium
Lake Geneva
whole
endosporecommunity
enriched
1.1
4.0
0.2
0.3
0.7
2.9
1.3
1.3
0.2
1.5
0.2
0.4
0.2
0.2
0.2
0.9
0.2
2.5
0.3
<0.1
1.4
0.5
0.1
0.3
0.2
0.1
0.4
0.2
0.4
18.3
0.2
4.6
6.3
<0.1
0.3
0.7
15.0
0.0
6.8
8.5
6.3
0.2
8.5
0.2
41.6
<0.1
33.2
0.1
Lake Baikal
whole
endosporecommunity
enriched
3.8
6.7
0.8
0.1
5.3
6.5
0.7
0.1
0.7
0.1
2.8
4.1
0.1
2.1
2.6
0.1
0.1
0.1
0.1
0.4
0.2
0.4
0.1
15.6
2.5
0.4
0.4
0.1
0.2
0.1
0.1
0.4
0.1
0.5
0.1
<0.1
0.1
0.3
2.5
0.5
3.3
2.6
0.3
0.1
1.7
47.9
0.1
4.3
0.2
<0.1
0.6
61.9
0.1
0.4
0.3
0.3
0.1
0.1
0.2
0.1
1.1
0.1
1.4
0.4
0.1
0.3
<0.1
3.6
0.5
0.9
0.2
0.4
1.3
0.4
1.1
0.2
2.0
1.7
4.8
0.4
0.1
0.6
0.1
0.1
0.4
0.4
<0.1
2.7
1.3
0.2
0.1
3.0
0.6
Ethanoligenens
Ruminococcus
Symbiobacteriaceae; Other
Symbiobacterium
Veillonellaceae; Other
BSV43
Pelosinus
[Acidaminobacteraceae]; Other
[Tissierellaceae]; Tepidimicrobium
o__OPB54
0.2
0.1
0.4
0.4
0.0
0.1
0.1
<0.1
0.4
2.1
0.5
<0.1
0.1
0.2
0.1
0.3
0.4
0.1
0.3
0.2
0.9
0.1
0.9
Supplementary Table 3. List of groups that disappeared when applying the treatment. These
results are based on the 16S rRNA sequencing data. Values correspond to abundance (%).
Differences between untreated and endospore-enriched samples (diff) of more than 1 % are
highlighted in bold.
Bacilli
Alicyclobacillaceae
Alicyclobacillus
Anoxybacillus
Marinibacillus
Paenibacillaceae
Cohnella
Paenibacillus
Lysinibacillus
Solibacillus
Ureibacillus
Thermoactinomycetaceae
Trichococcus
Clostridiales
Clostridiales;f__[Acidaminobacteraceae]
Clostridiaceae;g__02d06;s__
Alkaliphilus
Christensenellaceae
Caloramator
Oxobacter
Proteiniclasticum
Clostridiaceae;g__SMB53
Dehalobacterium
Gracilibacteraceae
Lachnospiraceae
Coprococcus
Epulopiscium
Pelotomaculum
Peptostreptococcaceae
Peptostreptococcaceae;g__[Clostridium]
Tepidibacter
Ruminococcaceae
Ruminococcaceae;g__Clostridium
Ethanoligenens
Ruminococcus
Symbiobacteriaceae
Symbiobacterium
Veillonellaceae
Clostridia;o__OPB54
untreated
Lake Geneva
endospore-enriched
0.22
1.53
0.44
0.22
0.87
0.05
1.43
0.15
0.10
0.18
-0.17
-0.10
-0.29
-0.12
-0.69
0.22
18.30
0.22
0.02
15.01
0.00
-0.20
-3.29
-0.22
0.22
0.03
diff
untreated
0.80
0.67
0.67
0.13
0.13
0.40
0.40
15.71
Lake Baikal
endospore-enriched
0.09
0.07
0.13
0.00
0.09
0.22
0.06
2.55
diff
-0.71
-0.59
-0.53
-0.13
-0.04
-0.17
-0.34
-13.17
0.13
0.11
-0.02
0.13
0.02
-0.11
0.27
0.13
0.19
0.04
-0.08
-0.10
1.60
0.13
0.54
0.13
-1.05
0.00
0.40
0.30
-0.10
0.27
0.11
-0.15
0.13
0.06
-0.08
0.40
0.13
-0.27
0.27
3.60
0.53
0.13
0.02
0.86
0.19
0.04
-0.25
-2.73
-0.35
-0.10
0.40
2.13
0.53
0.40
0.00
0.11
0.22
0.15
-0.40
-2.02
-0.31
-0.25
-0.19
0.22
41.61
0.44
0.02
33.22
0.00
-0.20
-8.39
-0.44
1.31
0.44
1.09
0.22
0.67
0.06
0.42
0.02
-0.64
-0.37
-0.67
-0.20
1.74
1.40
-0.35
4.79
2.97
-1.82
0.22
0.06
-0.15
0.44
0.44
0.03
0.19
-0.40
-0.24
Supplementary Table 4. List of groups that newly appeared when applying the treatment.
These results are based on the 16S rRNA sequencing data. Values correspond to relative
abundances (%). Differences between untreated and endospore-enriched samples (diff) of
more than 1 % are highlighted in bold.
1.09
0.22
0.65
Lake Geneva
endosporeenriched
3.98
0.32
2.97
0.00
0.00
0.00
1.31
0.02
0.02
0.02
3.80
+0.02
+0.02
+0.02
+2.49
0.00
1.31
0.00
0.00
0.00
0.02
1.98
0.03
0.03
0.02
+0.02
+0.67
+0.03
+0.03
+0.02
0.00
0.00
0.06
0.02
+0.06
+0.02
0.00
0.00
0.00
0.00
0.08
0.03
0.18
0.03
+0.08
+0.03
+0.18
+0.03
0.00
0.02
+0.02
untreated
Firmicutes
Bacilli
Bacillales
Bacillales;f__[Exiguobacteraceae]
Alicyclobacillaceae
Alicyclobacillus
Alicyclobacillus pomorum
Bacillaceae
Anaerobacillus
Anoxybacillus
Bacillus
Bacillus acidicola
Bacillus anthracis
Bacillus asahii
Bacillus badius
Bacillus cereus
Bacillus flexus
Bacillus horneckiae
Bacillus koreensis
Bacillus mannanilyticus
Bacillus muralis
Bacillus mycoides
Bacillus safensis
Bacillus thermoamylovorans
Geobacillus
Marinibacillus
Oceanobacillus
Oceanobacillus chironomi
Paenibacillaceae
Paenibacillus
Paenibacillus amylolyticus
Paenibacillus ginsengihumi
Paenibacillus illinoisensis
Thermobacillus composti
Planococcaceae
Lysinibacillus
Lysinibacillus massiliensis
Paenisporosarcina
Rummeliibacillus
Sporosarcina
Sporosarcina aquimarina
Ureibacillus
Pullulanibacillus
Thermoactinomycetaceae
Thermoactinomyces
Turicibacter
Clostridia
Clostridiales
Clostridiales;f__[Tissierellaceae]
Sedimentibacter
Tepidimicrobium
Caldicoprobacter
Christensenellaceae
diff
+2.89
+0.11
+2.32
0.00
1.09
0.00
0.00
0.00
0.00
0.22
0.34
1.18
0.02
0.02
0.02
0.05
0.50
+0.34
+0.10
+0.02
+0.02
+0.02
+0.05
+0.29
0.00
0.22
0.00
0.22
0.00
0.03
0.32
0.19
0.32
0.03
+0.03
+0.11
+0.19
+0.11
+0.03
0.00
0.00
0.00
0.44
0.02
0.05
0.26
0.68
+0.02
+0.05
+0.26
+0.25
0.00
0.00
0.00
0.00
0.03
0.03
0.15
0.02
+0.03
+0.03
+0.15
+0.02
3.73
Lake Baikal
endosporeenriched
6.65
5.19
0.00
6.44
0.07
+1.25
+0.07
2.80
0.00
4.08
0.02
+1.29
+0.02
1.73
2.32
+0.59
0.00
0.04
+0.04
0.00
0.00
0.00
0.00
0.04
0.02
0.04
0.04
+0.04
+0.02
+0.04
+0.04
0.00
0.02
+0.02
0.00
0.06
+0.06
0.00
0.00
0.02
0.09
+0.02
+0.09
0.00
0.00
0.02
0.02
+0.02
+0.02
0.40
0.13
0.00
0.00
0.41
0.19
0.06
0.07
+0.01
+0.05
+0.06
+0.07
0.40
0.00
0.00
0.00
0.51
0.04
0.02
0.02
+0.11
+0.04
+0.02
+0.02
0.27
2.40
0.49
3.30
+0.22
+0.90
0.00
0.02
+0.02
untreated
diff
+2.92
Clostridiaceae
Clostridiaceae;g__02d06
Clostridiaceae; Alkaliphilus
Caloramator
Clostridium
Clostridium acetobutylicum
Clostridium botulinum
Clostridium bowmanii
Clostridium butyricum
Clostridium celatum
Clostridium gasigenes
Clostridium intestinale
Clostridium ljungdahlii
Clostridium neonatale
Clostridium pasteurianum
Clostridium perfringens
Clostridium thermopalmarium
Clostridium tyrobutyricum
Oxobacter
Proteiniborus ethanoligenes
Sarcina
Eubacteriaceae
Gracilibacteraceae
Gracilibacter
Lachnospiraceae
Clostridium colinum
Clostridium fimetarium
Defluviitalea saccharophila
Roseburia
Roseburia faecis
Peptococcaceae
Desulfosporosinus
Desulfosporosinus meridiei
Desulfotomaculum aeronauticum
Peptostreptococcaceae
Peptostreptococcaceae; [Clostridium]
Peptostreptococcaceae; [Clostridium]
bifermentans
Peptostreptococcaceae; Clostridium
Peptostreptococcaceae; Clostridium
maritimum
Peptostreptococcaceae; Clostridium
ruminantium
Tepidibacter
Ruminococcaceae; Clostridium
Ruminococcaceae; Clostridium hungatei
Ethanoligenens
Oscillospira
Ruminococcus
Ruminococcus bromii
Symbiobacteriaceae
Veillonellaceae;g__BSV43
Pelosinus
Sporomusa
Halanaerobiaceae
Clostridia;o__MBA08
Anaerobrancaceae
Anaerobrancaceae;g__A55_D21
OPB54
Thermoanaerobacterales
4.58
6.32
0.00
0.00
3.05
6.83
8.52
0.02
0.18
4.63
+2.26
+2.20
+0.02
+0.18
+1.58
0.00
1.53
0.00
0.00
0.02
2.09
0.39
0.03
+0.02
+0.57
+0.39
+0.03
0.00
0.00
0.00
0.00
0.22
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.03
0.05
0.02
0.05
0.26
0.02
0.02
0.05
0.02
0.15
0.03
0.05
0.13
+0.03
+0.05
+0.02
+0.05
+0.04
+0.02
+0.02
+0.05
+0.02
+0.15
+0.03
+0.05
+0.13
0.00
0.00
0.00
0.00
0.00
0.02
0.06
0.05
0.02
0.03
+0.02
+0.06
+0.05
+0.02
+0.03
0.00
0.36
+0.36
1.96
2.69
+0.73
0.00
0.44
0.13
0.52
+0.13
+0.08
0.00
0.23
+0.23
0.44
0.00
0.00
0.22
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.87
0.00
0.50
0.18
0.08
0.31
0.05
0.02
0.05
0.02
0.02
0.05
0.29
0.02
0.03
0.03
0.05
0.02
0.89
0.02
+0.07
+0.18
+0.08
+0.09
+0.05
+0.02
+0.05
+0.02
+0.02
+0.05
+0.29
+0.02
+0.03
+0.03
+0.05
+0.02
+0.02
+0.02
2.66
4.33
+1.66
15.58
0.00
18.37
0.02
+2.79
+0.02
27.30
0.00
33.97
0.02
+6.67
+0.02
5.06
9.31
+4.25
0.00
0.13
+0.13
0.00
0.02
+0.02
0.00
0.13
0.02
0.22
+0.02
+0.09
0.00
0.02
+0.02
0.00
0.00
1.07
0.00
0.02
0.02
1.40
0.02
+0.02
+0.02
+0.34
+0.02
0.00
0.02
+0.02
0.00
0.02
+0.02
0.00
0.02
+0.02
0.00
0.00
0.09
0.28
+0.09
+0.28
Supplementary Materials and Methods
Strains and culture conditions
To test different cell disruption methods we selected the strains Escherichia coli (Gramnegative), grown in nutrient broth (NB); Paenibacillus alvei, Bacillus subtilis, and Bacillus
megaterium (Gram-positive endospore-forming Firmicutes), all grown in NB. All strains were
incubated at 30°C. Endospores were obtained by culturing at 37°C and vigorous shaking in
Schaeffer sporulation (Li et al.) medium (Schaeffer et al. 1965) for B. megaterium, and
modified versions of this medium (Leighton et al. 1971) (2 x SG for B. subtilis; 0.5 x SG for
P. alvei). Numbers of cells and endospores were determined microscopically using a
Neubauer counting chamber. All endospore preparations consisted of > 95% endospores. Prior
to the treatments, cells or endospores were recovered by centrifugation at 6000 xg for 5 min
and resuspended in 500 µl of physiological solution. A density of 108 cells or endospores per
milliliter were achieved, which corresponded to an absorbance of 0.3 A at 600 nm.
Sediment samples
Sediment samples were collected during a research campaign with the MIR manned
submersibles in Lake Baikal (Russia)(52° 53.00 N, 107° 10.00 E, at 1598.5 m depth) in
August 2010 and in Lake Geneva (Switzerland-France)(46° 27.03 N, 6° 42.52 E, at 284 m
depth) in June 2011. Sediment cores of approximately 30 cm length were retrieved using a
push-corer. Upon return to the surface, the fraction two-to-seven cm of the cores was
immediately sub-sampled in the center of the core as to avoid contamination using sterile cutopen syringes. Samples were then stored at -20°C until further processing.
Enrichment of endospores in sediment
The procedure to extract spores from sediment is schematically shown in Supplementary
Figure 1. In a first step, the biomass was separated from the sediment particles as described
elsewhere (Wunderlin et al. 2013). This method was validated with sediments amended with
endospores from P. alvei. To maximize the recovery of endospores, the procedure was carried
out as following: the slurry was then centrifuged at 20 x g for 60 s to remove coarse particles.
The supernatant (containing the cells) was collected on a nitrocellulose membrane of 0.2 µm
pore size and 47 mm diameter (Whatman, Dassel, Germany). The separation of biomass from
sediment was repeated for a second time on the same sample. The filter membrane was cut in
half and immediately frozen in liquid nitrogen and stored at -20°C. One half of the filter
membrane was kept as a control (untreated) and the other was subjected to the treatment for
destruction of vegetative cells. The filter half was transferred into a centrifuge tube with 900
µl tris-EDTA buffer and gently dispersed by vortexing. The mix was then incubated in a water
bath at 65°C for 20 min and cooled at 4°C for 5 min. Lysozyme was added to the sample in a
final concentration of 2 mg/ml and incubated at 37°C for 60 min and 120 rpm shaking. Then
250 µl of NaOH and 250 µl of SDS were added to obtain final concentrations of 0.5 N and
1%, respectively. The mix was then incubated at room temperature for 60 min with slow
agitation (100 rpm). The sample was afterwards filtered through 0.2 µm pore size
nitrocellulose membrane of 17 mm diameter with a vacuum pump and washed with sterile
physiological solution to remove residual detergent. The vacuum pump was turned off for
subsequent DNAse treatment directly on the filter membrane. The mix of 400 µl containing
water, 1 x reaction buffer and 2 µl DNase (New England Biolabs, Ipswich, MA, USA) was
added directly onto the membrane and left standing for 15 min to digest the extracellular DNA
in the sample. Then the sample was washed with physiological solution by applying the
vacuum again. Once dried, the filter membrane was stored at -20°C until DNA extraction.
Quantification of gene copy numbers
Quantification of bacterial DNA in sediment extracts was carried out by real-time quantitative
PCR of the V3 region of the 16S rRNA gene with the primers 338f and 520r (Ovreas et al.
1997). The detailed protocol is described in Wunderlin (Wunderlin et al. 2013). The qPCR
mix contained 0.5 ng DNA template. All extracts were analyzed in triplicates. For
quantification three independent plasmid standards series with 300 to 3,000,000 gene
copies/µL of the 16S rRNA gene of an environmental clone were included.
Quantification of spo0A gene was done as mentioned in (Bueche et al. 2013) but with TaKaRa
SYBR Premix Ex Taq II (TaKaRa, Shiga, Japan) with 1.4 or 0.5 ng DNA as template. The
reaction mix was composed of 1 x TaKaRa Premix, 0.75 µM of forward and 0.45 µM of
reverse primer and water to total volume of 20 µl. Three independent plasmid standards series
with 30 to 300,000 gene copies/µL of spo0A gene of B. subtilis were included. Quantification
of the rpoB gene was done with 0.5 ng DNA template, 0.3 µM of each primer (Dahllöf et al.
2000) and 1 x TaKaRa Premix in a total reaction volume of 20 µl. All qPCR reactions were
run with a Rotor-GeneTM 6000 instrument (QIAGEN, Hilden, Germany) with the program:
enzyme activation at 95°C for 5 min, 40 cycles of denaturation at 95°C for 5 s, annealing at
55°C for 20 s and extension at 72°C for 30 s. Thresholds (Th), Ct values, and derivatives of
melting curves were determined using Rotor-Gene 6 software (QIAGEN).
Quantification of endospores via detection of dipicholynic acid
Quantification of dipicholynic acid (DPA) in sediment was done by measuring the
fluorescence emitted by a terbium-DPA complex as described previously for soil (Brandes
Ammann et al. 2011). Briefly 0.25 g of freeze-dried sediment was inserted in glass screw-cap
tubes and 4.3 ml Na-acetate buffer (0.2 M, pH 5) and 480 µl of AlCl3 (0.5 M) were added and
mixed with the sediment. Screw-caps were closed and samples were autoclaved at 121 °C for
20 minutes. After cooling down, the samples were centrifuged at 4000 x g for 3 min. One ml
of supernatant was then mixed with 1 ml of TbCl3 (30 µM) and put to quartz cuvette for
fluorescence measurement with a Perkin-Elmer LS50B fluorometer. The excitation was set to
272 nm with a silt of 2.5 nm and emission was measured at 545 nm (silt 2.5 nm) with a delay
of 50 us. The signal was integrated over 1.2 ms of measurement. Five such measurements
were taken and mean value was calculated. All quantifications were done with three
independent replicas. The values fluorescence values were converted to DPA concentration
using an 8 point standard curve from 0.05 µM to 10 µM DPA.
Endospore solutions of B. subtilis and P. alvei (> 95 % endospores) were quantified
microscopically using a cytometer with Neubauer improved ruling. Two spore solutions of 1 x
108 endospores ml-1 was taken for each strain and DPA was quantified as above. The average
DPA concentration of the two strains (1.0 ± 0.2 x 10-8 µM per endospore) was taken for the
estimation of endospore numbers in sediments.
Amplicon sequencing
DNA yield in the treated samples was too low for direct amplicon sequencing. Therefore all
samples were amplified with the primers Eub9-27f and Eub1542r to produce a 1,533 bp
fragment of the 16S rRNA gene (Liesack et al. 1991). PCR reactions were performed with 0.5
ng DNA template in 1 x reaction buffer (TaKaRa), 2 mM MgCl2, 10 µg bovine serum
albumin (BSA; New England Biolabs), 1 U of Ex Taq Polymerase (Proof-reading polymerase;
TaKaRa), 200 µM of each dNTP and 200 nM of each primer in a total reaction volume of 50
µl, completed with PCR-grade water. Negative controls (1 µl PCR-grade water) and positive
controls (1 ng P. alvei DNA template) were included in all reactions. Reactions were done
with an Arktik Thermo Cycler (Thermo Fisher Scientific, Vantaa, Finland) with the following
temperature program: initial denaturation at 94°C for 2 min; then 10 cycles of denaturation at
94°C for 30 s, touchdown annealing starting at 60°C with decrease of 0.5°C per cycle for 45 s
and elongation at 72°C for 1 min; followed by 30 cycles of denaturation at 94°C for 30 s,
annealing at 55°C for 45 s and elongation at 72°C for 1 min; and a final extension at 72°C for
5 min. Duplicate reactions (total volume of 100 µl) were then pooled and purified with
MultiScreen PCRµ96 plate (Merck Millipore, Darmstadt, Germany). Re-eluted sample in 20
µl were then loaded onto a 1% agarose electrophoresis gel and run for 40 min at 80 V. Gel
bands of 1,533 bp size were excised and purified with QiaQuick Gel extraction kit
(QIAGEN). A minimum of 500 ng of amplified DNA was then sent for barcode amplicon
sequencing with Roche GS FLX+ (Eurofins MWG Operon, Ebersberg, Germany).
Sequence analysis of 16S rRNA gene data
Sequences were analysed using QIIME for high-throughput 16S rRNA sequences (Caporaso
et al. 2010). After quality filtering (minimum quality score of 25), the sequences were denoised and checked for chimera with the usearch_qf.py program using the usearch method
(Edgar et al. 2011). The filtered sequences were then clustered de novo into putative OTUs
(identity of >97%) with the pick-otus.py program using the Uclust method (Edgar 2010).
Taxonomy was assigned with the RDP classifier (Wang et al. 2007). All metagenomic
sequences were submitted to Sequence Read Archive (SRA) under BioProject number
PRJNA214154 (BioSamples SRS431098 (Lake Geneva) and SRS431097 (Lake Baikal)) and
accession numbers SRR1011310, SRR1011311, SRR1011312, SRR1011313, SRR1011314,
SRR1011315, SRR1011316, SRR1011317.
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