Materials and methods
Sampling
Using a remotely operated underwater vehicle (ROV) equipped with a hydraulic
pump device, we collected samples of microbial mats from a actively venting
hydrothermal chimney at a depth of approximately 2350 meters at the Loki’s
Castle vent field (73°30´ N and 8°E) in June 2009. The samples were transported
through the water column at ambient temperature (-0.7 °C – 2 °C) for
approximately 2 hours. After arrival on the ship, the samples were kept on ice
for less than 30 minutes before they were frozen in liquid nitrogen and stored at
-80 °C until further processing. The sampling device was rinsed with sterile
seawater between subsequent dives. Further processing of the samples,
designated BSROV3 and BSROV4, were done in an onshore laboratory.
DNA and RNA extraction
Total DNA was extracted from the BSROV4 sample using the FastDNA spinkit for
soil (MP Biomedicals). Total DNA and RNA from the BSROV3 was extracted from
the same starting material using RNA power soil total RNA isolation kit and RNA
power soil DNA elution accessory kit (MoBio Laboratories Inc.). As suggested by
the manufacturer, we used a Phenol:Chloroform:Isoamyl-alcohol solution with
pH 6.6 (Ambion).
Amplification of 16S rRNA genes was performed as previously described
(Roalkvam et al., 2011) using a two-step PCR approach. The first PCR step was
run with less than 20 ng template in 25 cycle reactions using primers universal
for Archaea and Bacteria - Un787f (50-ATTAGATACCCNGGTAG) (Roesch et al.,
2007) and Un1392r (50-ACGGGCGGTGWGTRC) (modified from Lane et al.,
1985). In order to minimize PCR bias, reactions were run in triplicates. The
triplicate PCR products were pooled and rinsed with the MinElute PCR
purification kit (Quiagen). In the second PCR step, approximately 20 ng PCR
product from the first PCR step was used as template in a 5 cycle PCR reaction,
using the same primers as used in the first step except that a sample specific MID
sequence, and a GX FLX Titanium primer A was attached to the forward primer
and GS FLX Titanium primer B was attached to the reverse primer. All PCR
reactions were run in 25 µl reactions. PCR product quantification, rinsing of PCR
products after the second PCR step and sequencing were performed as
previously described (Roalkvam et al., 2011).
cDNA synthesis
The extracted RNA was treated with RNase-Free DNase (Promega) in order to
remove any residual DNA. Conversion of RNA to cDNA was performed in
triplicate via random hexamer priming and reverse transcription by applying the
Superscript Double-Stranded cDNA synthesis Kit (Invitrogen) using the
manufacturer’s protocol. The triplicate cDNA conversions were pooled and
concentrated by vacuumcentrifugation (Eppendorf Concentrator 5301) to obtain
10 µl cDNA with a concentration of 119 ng/µl. The cDNA concentration was
determined by SYBR-Green staining as previously described (Roalkvam et al.,
2011). A total of 949 µg of double stranded cDNA was subjected to 454pyrosequencing at the Norwegian Sequencing Centre
(http://www.sequencing.uio.no/).
Sequence filtering and extraction of rRNA reads
Removal of sequencing noise, PCR point error and chimeric sequences from the
amplicon reads was performed using AMPLICONNOISE (Quince et al., 2011).
Filtering of the cDNA reads was done in MOTHUR (Schloss et al., 2009) by
removing sequences with one or more ambiguous bases, or which had an
average quality score below 25. The filtered cDNA reads were compared to SSU
and LSU rRNA gene sequences retrieved from the National Center for
Biotechnological Information (NCBI) (http://www.ncbi.nlm.nih.gov/), using
BLASTN (Altschul et al., 1997). Reads with a bitscore above 50 to any rRNA gene
were regarded as derived from rRNA and extracted from the dataset by use of in
house Perl scripts.
OTU clustering, rarefaction and calculation of diversity indices
The amplicon reads from both samples were concatenated and clustered in
operational taxonomic units (OTUs) using AMPLICONNOISE (Quince et al., 2011)
with the maximum linkage clustering algorithm and a 3 % cutoff. Chao1
estimates with confidence intervals were calculated using MOTHUR (Schloss et
al., 2009). Other diversity indices and data for construction of rarefaction curves
were calculated using the Vegan package in R.
Taxonomic and functional analyses
For taxonomic analyses of amplicon and rRNA derived cDNA reads, sequences
were compared to the modified version of the Silva SSURef release 104 (Pruesse
et al., 2007; Lanzén et al., 2011), using BLASTN. In this modified version the
taxonomy within several taxa, including the Epsilonproteobacteria, is manually
reviewed and edited (Lanzén et al., 2011). Non-rRNA derived cDNA sequences
were compared to protein coding sequences in NCBI using BLASTX. BLASTN and
BLASTX result files were uploaded to the MEGAN software (version 4.62.2)
(Huson et al., 2011), which assigns reads to taxa using the last common ancestor
(LCA) algorithm. For amplicon reads and rRNA derived cDNA reads we used a
MEGAN bitscore threshold of 150 whereas for protein coding reads the MEGAN
bitscore threshold was 50. In all analyses in MEGAN the minimum support
parameter was set to 1. Otherwise the default parameters were used. Functional
assignments of non-rRNA reads were done with MG-RAST (Meyer et al., 2008)
and with BLASTX. The BLASTX searches were based on top hits and a bitscore
treshold of 50. The database used in BLASTX searches was either NCBIs RefSeq
protein or proteins from Sulfurovum sp. NBC37-1 only. The analyses in MG-RAST
were performed using an e-value cutoff of 10-5 in searches against the SEED
subsystems database.
Phylogenetic analyses
Phylogenetic analyses were performed in ARB (Ludwig et al., 2004) using the
following approach: First, nearly full length 16S rRNA gene sequences (E.coli
positions 28-1392) were used to make a backbone tree by applying the
Neighbor-Joining algorithm (Saitou and Nei, 1987) with the Jukes-Cantor
correction (Jukes and Cantor, 1969). Shorter sequences (OTU representatives
from the amplicon datasets) were added to the tree using ARBs quick add option.
The phylogenetic tree was constructed by applying a bacterial positional
variability filter (pos_var_Bacteria_102).
Deposition of sequence data
All sequence data are publically available as raw data (sff files) in the Sequence
Read Archive (SRA, http://www.ncbi.nlm.nih.gov/sra) under the accession
number SRA051260. Non-rRNA sequences are publically available from MGRAST (http://metagenomics.anl.gov/) under the accession number 4460441.3. A
fasta file of one representative sequence of each OTU constructed from the 16S
rRNA gene amplicon analyses is available as supplementary material.
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