Supplementary Materials
Materials and Methods
Analysis of terminal restriction fragment length polymorphism data
The abundance of individual terminal restriction fragments (TRFs) was calculated as
previously outlined (Clement et al., 1998; Enright et al., 2007) except that TRFs
representing <5% of the total area of respective restriction patterns were excluded
from further analyses. Terminal restriction fragment length polymorphism (TRFLP)
analysis could subsequently be successfully applied to monitor the fate of specific
clones throughout the trial, taking into consideration that discrepancies of ± 2 to 3
base-pairs (bp) have to be taken into account when comparing predicted and measured
TRFs (Liu et al., 1997; Kitts, 2001; Lueders and Friedrich, 2003). The identity of
TRFs was assigned through (i) performing in silico enzyme restrictions of clone
library sequences (ii) the TAP-TRFLP tool of the Ribosomal Database Project
database (Marsh et al., 2000) and (iii) an in-house database of simulated archaeal and
bacterial in silico restrictions (Collins et al., 2003; McHugh et al., 2003; McHugh et
al., 2004; Enright et al., 2007, 2009). TRFLP data analysis was conducted either
qualitatively, by creating binary matrices whereby the presence (‘1’) or absence (‘0’)
of individual TRFs was scored, or semi-quantitatively, by calculating the relative
abundance of TRFs normalised by the total area of the respective TRF patterns.
Cluster analysis was performed using those matrices and a Multivariant Statistical
Package (MVSP; Kovach, 1999). Dendrograms were constructed using unweighted
pair-group methods using arithmetic averages (UPGMA) analysis and Jaccard
distance measure. Ordination by non-metric multidimensional scaling (NMS; Kruskal,
1964) was also performed based on the presence/absence matrices to track the
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temporal changes in community structure. NMS condenses a data set to a point on the
resulting plot without loss of information so that the communities with similar TRFLP
profiles will be plotted close together (McCune and Grace, 2002). In this study, the
NMS ordination was carried out based on Sorensen (Bray-Curtis) distance measure
with PC-ORD software (version 5.0; Grandin, 2006).
Quantitative polymerase chain reaction analysis
Quantitative polymerase chain reaction amplifications were performed in a two-step
thermal cycling: pre-denaturation for 10 min at 94○C, followed by 40 cycles of 10 s at
94○C and 30 s at 60○C. All templates were analysed in triplicate. The quantification
standard curves were constructed as previously described (Yu et al., 2006) using
representative strains of the target groups: Methanospirillum hungatei JF1 (DSM 864)
and Methanomicrobium mobile BP (DSM 1539) for MMB set and Methanosaeta
concilli GP6 (DSM 3671) for Mst-set. Three custom recombinant plasmids containing
the full-length 16S rRNA gene sequences of the representative strains were used as
the templates for constructing standard curves. For MMB-set, the equimolar mixture
of its two different standard plasmids was used. The mass concentrations of plasmids
were measured in duplicate using a Qubit system (Invitrogen, U.S.A.) and converted
to their copy concentrations (Yu et al., 2006). A 10-fold dilution series of 101 to 109
copies l-1 was generated for each standard solution and analysed in triplicate by
qPCR. The threshold cycle values (CT) determined were plotted against the logarithm
of their template copy concentrations. The 16S rRNA gene copy concentrations of
target groups were determined against the corresponding standard curves within the
linear range (r2 >0.995).
2
Table SM1. Archaeal clone library analysis; In silico TRFLP restrictions of clonal sequences.
Library
Accession No.
Coverage
(%)
Lineage1
Seed
DQ679927
DQ679928
DQ679929
DQ679930
DQ679931
DQ679932
DQ679933
13
3
3
10
7
13
51
Uncultured Crenarchaeota
Uncultured Crenarchaeota
Methanosaeta
Methanosaeta
Methanomethylovorans
Methanosaeta
Methanosaeta
Sequence Homology TRFLP in silico restriction length3
(%)2
HhaI-forward
HhaI-reverse
99
n.d.
231
99
n.d.
231
99
n.d.
577
99
n.d.
582
97
175
n.d.
98
193
n.d.
99
193
n.d.
Day 1,228
FJ347527
FJ347528
FJ347529
FJ347530
FJ347531
FJ347532
43
19
30
4
3
1
Methanosaeta
Methanocorpusculum
Methanocorpusculum
Methanocorpusculum
Methanosaeta
Methanocorpusculum
99
97
99
99
94
98
1
Based on Ribosomal Database Project classification.
Based on National Centre for Biomedical Information classification.
3
In base-pairs.
n.d. - not determined.
2
3
195
326
327
326
193
327
581
122
122
122
582
364
Figure SM1. Non metric multidimensional scaling (NMS) plot for the TRFLP spectra
derived from (a) bacterial and (b) archaeal HhaI-reverse primer profiles. Percentage of
variance for each axis is given in parenthesis.
(a)
(b)
4
Figure SM2. Cluster analysis (UPGMA) of bacterial terminal restriction fragments
resolved using (a) HhaI-reverse and (b) HhaI-forward primer profiles.
(a)
(b)
5
Figure SM3. Cluster analysis (UPGMA) of archaeal terminal restriction fragments
resolved using (a) HhaI-reverse and (b) HhaI-forward primer profiles.
(a)
(b)
6
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