Download Supplementary Tables. Table S1. RNA extraction details and primer

Supplementary Tables.
Table S1. RNA extraction details and primer combinations used for 16S rRNA, mcrA
mRNA, and dsrAB mRNA clone libraries for all the cores.
Core
Depth
(cmbsf)
16S rRNA
Dec 06
24-27
Jul 05-2
54-57
Jul 05-2
54-57
Jul 08-2
48-51
Jul 05-1
60-63
Dec 06
69-72
Jul 05-2
87-90
Jul 05-2
87-90
Jul 05-2
87-90
Jul
Jul
Jul
Jul
Jul
Jul
Jul
Jul
Jul
mcrA mRNA
08-2
3-6
08-2
9-12
08-2
18-21
05-2
54-57
08-2
48-51
05-1
60-63
05-1
87-90
05-1
87-90
05-2
87-90
RT-PCR #
combined
for
sequencing
Weight
(g) or
volume
(ml) of
sample
Method of
RNA
extraction
Primersa
#
clones
A8f-A915r
A8f-A1492r/A8f-A1492r
A8f-A1492r/A21f-A915r
A8f-A915r
A8f-A1492r/A21f-A915r
A8f-A915r
A21f-EelMS932r
A21f-A915r
A8f-A915r
52
30
61
33
68
90
30
25
43
1
3
1
1
1
1
3
1
3
2.78 g
4.5 ml
4.5 ml
2.06 g
4 ml
4.96 g
3 ml
3 ml
3 ml
Phenol/BB
Phenol/BB
Phenol/BB
MoBio PS
Phenol/BB
Phenol/BB
Phenol/BB
Phenol/BB
Phenol/BB
ANME-1 mcrf/r
ANME-1 mcrf/r
ANME-1 mcrf/r
ME1-2/ME1-2
ANME-1 mcrf/r
ME1-MCRr/MCRf-ME2
ME1-2/ME1-2
MCRf-r/MCRf-r
ME1-2/ME1-2
12
26
30
18
36
43
2
37
3
1
1
1
3
1
3
3
3
3
2.18 g
1.98 g
1.98 g
4.5 ml
2.06 g
4 ml
3 ml
3 ml
3 ml
MoBio PS
MoBio PS
MoBio PS
Phenol/BB
MoBio PS
Phenol/BB
Phenol/BB
Phenol/BB
Phenol/BB
b
c
dsrAB mRNA
Dec 06
24-27
Dsr1f-4r/1f1-1r1
32
2
2.78 g
Phenol/BB
Jul 05-2
51-54
Dsr1f-4r/1f1-1r1
79
2
3.44 g
Phenol/BB
Jul 05-1
60-63
Dsr1f-4r/1f1-1r1
7
3
3.44 g
Phenol/BB
Jul 05-1
84-87
Dsr1f-4r/1f1-1r1
29
1
4.70 g
Phenol/BB
a
See Table S2 for primer sequences. When two primer sets are listed, the first was used for initial RT-PCR
and the second for nested PCR.
b
Some samples had multiple identical and parallel RT-PCR amplifications combined for sequencing.
c
Phenol/BB stands for the phenol and bead-beating method as described previously (1) and Mobio PS
stands for the MoBio RNA PowerSoil kit, used as described in its instructions.
Table S2. Primers used for reverse transcription and amplification for cDNA clone
libraries
Primer
Target
gene
Predicted
target group
Sequence (5’ to 3’)a
Archaea
Archaea
Archaea
Archaea
ANME-2
Sulfate reducers
Sulfate reducers
Sulfate reducers
Sulfate reducers
Methanogens
Methanogens
Methanogens
Methanogens
ANME-1
ANME-1
TCC GGT TGA TCC TGC C
TTC CGG TTG ATC CYG CCG GA
GTG CTC CCC CGC CAA TTC CT
GGC TAC CTT GTT ACG ACT T
AGC TCC ACC CGT TGT AGT
ACS CAY TGG AAG CAC G
GTG TAG CAG TTA CCG CA
CAG GAY GAR CTK CAC CG
CCC TGG GTR TGR AYR AT
TAY GAY CAR ATH TGG YT
ACR TTC ATN GCR TAR TT
GCM ATG CAR ATH GGW ATG TC
TCA TKG CRT AGT TDG GRT AGT
GAC CAG TTG TGG TTC GGA AC
ATC TCG AAT GGC ATT CCC TC
A8f
A21f
A915r
A1492r
EelMS-932r
Dsr1f
Dsr4r
1f1
1r1
MCRfb
MCRr
ME1b
ME2b
ANME1-mcr1
ANME1-mcr2
16S
16S
16S
16S
16S
dsrA
dsrB
dsrA
dsrB
mcrA
mcrA
mcrA
mcrA
mcrA
mcrA
CARD-FISH
Probe
Formamide
concentration
(%)
60
60
Predicted
target group
Sequence (5’ to 3’)
ANME-1
ANME-2
Bacteria
55
60
Archaea
Putative AOM
syntrophic SRB
AGT TTT CGC GCC TGA TGC
AGC TCC ACC CGT TGT AGT
GCA GCC ACC CGT AGG TGT and
GCT GCC ACC CGT AGG TGT and
GCT GCC TCC CGT AGG AGT
GTG CTC CCC CGC CAA TTC CT
TCC ACT TCC CTC TCC CAT
Target
gene
Predicted
target group
Sequence (5’ to 3’)
ANME-1
GCT TTC AGG GAA TAC TGC
ANME1-350
EelMS932
Eub I-III
Arc915
DSS 658
qPCR/RTqPCR
primer
ANME1-628f
16S
Anneal
temp
(°C)
50 or 58
50
50 or 58
55
59
54
54
48
48
50
50
55
55
58
58
Ref
Hybridization
temp
(°C)
Ref
35
35
35
(4)
(4)
(10)
35
35
(11)
(12)
Anneal
temp
(°C)
60
Ref
(2)
(3)
(3)
(2)
(4)
(5)
(5)
(6)
(6)
(7)
(7)
(8)
(8)
(9)
(9)
This
study
(4)
ANME1-830r
16S
ANME-1
TCG CAG TAA TGC CAA CAC
60
a
Y = C/T; R = A/G; M = A/C; W = A/T; K = G/T; H = A/C/T; D = A/G/T
b
All 5 ANME-1 fosmids containing mcrA from (Hallam et al, 2003)(13) had 4 mismatches to ME1, 2
mismatches to ME2, 2 mismatches to mcrF, and 0 mismatches to mcrR. Therefore, lack of amplification
with ME1, ME2, or mcrF primers is not good evidence of the absence of ANME-1 mRNA.
Supplementary Figures.
Supplemental Figure S1. Neighbor-joining tree of methanogen-related archaeal 16S
rRNA transcript sequences, with distance- or parsimony-based bootstrap support (>60%)
from 1000 repetitions listed at the nodes, respectively, excluding the poorly aligned helix
6. Numbers of clones represented by each 97% OTU group within each core and depth
are shown in parentheses. Cores are named A for Jul 05-1, B for Jul 05-2, E for Dec 06,
and G for Jul 08-2. Clones from the AOM zone are in red, clones from the
methanogenesis zone are in blue, and tree was rooted with Methanocaldococcus
jannaschii. Scale bar represents 10% distance.
Supplemental Figure S2. Neighbor-joining trees of amino acid translations of dsrAB
mRNA cDNA following the groupings of Leloup et al. (2007) (14) and Zverlov et al.
(2005) (15), with distance- and then parsimony-based bootstrap support (>60%) from
1000 repetitions listed at the nodes. Numbers of clones represented by each 97% OTU
group (determined from nucleic acids) are shown in parentheses. Clones from the AOM
zone are in red, clones from the methanogenesis zone are in blue, with naming
conventions as for Figure S1.
Supplemental Figure S3. Percentage of positive RT-PCR amplification attempts for
mcrA mRNA (using only general mcrA primers, excluding the ANME-1 mcrf/r which are
specific for ANME-1) and dsrAB mRNA within the AOM (red) and the methanogenic
(blue) zones for cores Jul 05-1, Jul 05-2, Dec 06, and Jul 08-2. Values are significantly
different between AOM and methanogenesis zones (p > 99% for mcrA, p > 98% for
dsrA) with a two-tailed chi-squared test, and the numbers written on the bars are the total
numbers of amplification attempts. Only amplification attempts with primers and RTPCR conditions that had previously been shown to result in a positive amplification were
included.
Supplemental Figure S4. Map of the coast of North Carolina and the White Oak River,
near Beaufort, North Carolina. Inset shows the location of station H as the river opens up
into brackish estuarine sediments. Map made using the free Online Map Creation tool at
http://www.aquarius.ifm-geomar.de.
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