Supporting Information for:
Development and application of primers for the class Dehalococcoidia (phylum Chloroflexi)
enables deep insights into diversity and stratification of subgroups in the
marine subsurface
Kenneth Wasmund1,5, Camelia Algora1, Josefine Müller1, Martin Krüger2, Karen G. Lloyd3,6,
Richard Reinhardt4, Lorenz Adrian1
1
Helmholtz Centre for Environmental Research – UFZ, Permoserstraße 15, D-04318 Leipzig,
Germany.
2
Federal Institute for Geosciences and Natural Resources (BGR), Stilleweg 2, D-30655
Hannover, Germany.
3
Center for Geomicrobiology, Department of Bioscience, Aarhus University, Ny Munkegade
114, DK-8000 Aarhus C, Denmark.
4
Max Planck Genome Centre Cologne, Carl-von-Linné-Weg 10, D-50829, Cologne, Germany.
5
Current address: Division of Microbial Ecology, Faculty of Life Sciences, University of Vienna,
Vienna, Austria.
6
Current address: Department of Microbiology, University of Tennessee, Knoxville, TN, USA.
Corresponding Author: Kenneth Wasmund
E-mail: kwasmund@gmail.com
Aarhus Bay cores sampling and biogeochemical measurements
Two 3 meter gravity cores were obtained on the R/V Tyra from the ‘Mimosa’ site in
Aarhus Bay (56°09.604’ N, 10°28.104’ E) in 16.3 m water depth. Core A was obtained March
22, 2011, with bottom water temperature 2.5°C. Core B was obtained November 22, 2011. All
sampling was done in a 4°C cold room at Aarhus University, and samples used for molecular
biology were frozen immediately at -80°C. Methane was subsampled by drilling holes into the
core liner and removing 4 ml sediment plugs which were placed immediately into 4 ml 1 M KOH
in a 20 ml serum vial, which were capped and then frozen. Methane measurements were made on
headspace gas by using a gas chromatograph with flame ionization detector (SRI Instruments).
For sulphate measurements on Core A, porewater was removed via rhizones attached to an
evacuated glass serum vial. For sulphate measurements on Core B, plastic 10 ml tubes were filled
with sediment and centrifuged at 5000 rpm for 10 minutes. Resulting porewater was then filtered
into 1.5 ml plastic tubes and measured six days later. Sulphate was measured for both cores by an
ion chromatograph (Dionex).
Total organic carbon and total carbon measurements for Baffin Bay cores
For measurements of total organic carbon (TOC) in Baffin Bay cores (Algora et al.,
2013), 1 g of dry and homogenized sediment was measured in a LECO RC-412 carbon
determinator (LECO Corporation, MI, USA). The temperature program consisted of two phases:
phase 1 for the measurement of TOC with parameters of 80°C min-1 from 100°C to 530°C and
phase 2 for the measurement of total inorganic carbon with parameters of 100°C min-1 from
530°C to 1000°C. All values are reported as mean of duplicate measurements in weight
percentages.
Design and testing of DEH specific PCR primers
Primers were designed to cover as many DEH 16S rRNA gene sequences as possible and
to have a minimum of non-target sequence hits to other bacterial groups. The high sequence
diversity within published DEH 16S rRNA gene sequences meant that it was impossible to
design primers that covered every DEH 16S rRNA gene sequence without also matching nontarget sequences. Nevertheless, alignment of the developed primer sequences with 482 random
DEH sequences from our database showed that the primers had high overall coverage (Tables S1
and S2). For instance, the lowest coverage for any nucleotide positions towards the critical 3’
ends of either primer was 93%. This was deemed as a good compromise for keeping one primer
set that covered most of the diversity versus using multiple primer sets, for simplicity and to
enable consistent comparisons among various samples using approaches such as quantitative
real-time PCR.
During initial examination of primer specificity as determined by cloning and sequencing
of PCR products, non-target sequences were also identified that were not predicted by using ARB
alone. To increase the specificity of the assay, all DEH sequences were exported from the ARB
database and aligned with the non-target sequences amplified with the early versions of the
primers using the alignment tool MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/). Using
these alignments, primers were specifically manipulated towards the 3’ ends of the primers to
reduce the chances for the possible amplification of non-target sequences. By doing this, a
specific primer pair that matched the vast majority of DEH sequences exported from the database
was identified. Considering these improved primers also did not enable entirely specific
amplification during initial testing, the specificity was further increased by precisely optimizing
annealing temperatures using clones containing DEH or non-target sequences containing known
mismatches to the primers. This enabled precise determination of annealing temperatures that
could distinguish target from non-target sequences.
The specificity of the primers was also evaluated by classification of all quality controlled
sequences generated by pyrosequencing. The specificity was evaluated prior to bioinformatic
removal of non-target sequences, which was used as a later step to ensure the diversity and
community structures of DEH were examined specifically. This analysis revealed high overall
specificity, with 97% of all quality controlled sequences from all samples (total no. 43920) being
classified as DEH. Examination of the classification of pyrosequencing data into DEH subgroups showed that the primers had the ability to amplify a diverse range of DEH 16S rRNA
gene sequences spanning all sub-groups from the various samples analysed (Fig. 2 in Main text).
Clone library construction and Sanger DNA sequencing
To initially examine and optimize the specificity of the primers and real-time PCR assays,
several samples were analysed by cloning and Sanger sequencing. This was conducted using
DNA sourced from various marine and terrestrial samples (listed in Table 1 of the main text), and
also served to exemplify the ability to amplify a diverse range of DEH sequences from the
environment (Fig. S2). For this, each sample was amplified by PCR in three independent
replicate reactions, amplicons from the replicates were pooled, and the pooled amplicons were
cloned into the pGEM-T-Vector (Promega) according to the manufacturers instructions and
transformed into NEB 5-alpha Competent E. coli (High Efficiency) (New England Biolabs). PCR
products of the inserts derived from randomly picked individual colonies were sent to Macrogen
Inc. (Amsterdam, Netherlands) for purification and sequencing with an ABI3730 XL automatic
DNA sequencer (Sanger sequencing), using the vector specific M13f primer.
Phylogenetic analysis of 16S rRNA genes determined by Sanger sequencing
Sequences of 16S rRNA genes determined by Sanger sequencing were initially checked
for quality using Chromas Lite software version 2.01 (Technelysium) before being truncated to
exclude primer and vector sequences. 16S rRNA gene sequences were then aligned to the preexisting SILVA-based alignment described in the Experimental procedures of the main text (used
for
phylogenetic
analyses
and
sub-group
deliniations)
using
the
‘Add unaligned
sequence(s) to existing alignment’ function in the online version of MAFFT (version 7) (Katoh
and Standley, 2013). This alignment was imported into ARB, a phylogenetic tree was constructed
using the Randomized Axelerated Maximum Likelihood algorithm implemented in ARB
(RAxML vesion 7.0.3), and Sanger sequences of approximately 475 bp were inserted into the
tree (Fig. S2).
References
Algora, C., Gründger, F., Adrian, L., Damm, V., Richnow, H.-H., and Krüger, M. (2013)
Geochemistry and microbial populations in sediments of the Northern Baffin Bay, Arctic.
Geomicrobiol J 30: 690-705.
Katoh, K., and Standley, D.M. (2013) MAFFT multiple sequence alignment software version 7:
improvements in performance and usability. Mol Biol Evol 30: 772-780.