STRAIN-LEVEL GENOMIC VARIATION IN NATURAL POPULATIONS OF LEBETIMONAS
FROM AN ERUPTING DEEP-SEA VOLCANO
Julie L Meyer and Julie A Huber
Josephine Bay Paul Center, Marine Biological Laboratory, Woods Hole, MA, USA
Supplementary Material:
Growth of Lebetimonas under different conditions
The enrichment media consisted of anaerobic saltwater with H2 as an electron donor,
elemental sulfur as an electron acceptor, ammonia as a nitrogen source, CO2 as a carbon source,
trace elements, vitamins, and 0.1g/L yeast extract. All six isolated strains grew with or without
the low level of yeast extract used in the enrichment media, but did not grow without vitamins.
Lebetimonas strain JS085 was transferred to media without yeast extract prior to testing the
following alternative electron donor/acceptor pairs: H2/NO3, formate/S0, formate/NO3,
thiosulfate/NO3, and thiosulfate/O2. Growth of Lebetimonas strain JS085 was evaluated at pH
levels of 5, 5.5, 6, 6.5, 7, 7.5, 8, and 8.5 and at temperatures from 25°C to 70°C, at 5°C intervals.
Lebetimonas strain JS085 could not use thiosulfate as an electron donor nor use nitrate or oxygen
as an electron acceptor under the conditions tested. Growth in strain JS085 occurred at pH 6 to 8
and at temperatures from 40°C to 60°C. Cultures grown with the original enrichment media at
55°C became turbid within 24 hours, with a doubling time of 3 hours in strain JS085 under these
conditions. Cultures without yeast extract grew much slower, taking up to a week to become
turbid. Cells were motile, short, curved rods, about 1 µm in length.
Genome assembly
Prior to assembly, Illumina sequences were quality filtered using adaptive window
trimming and a quality threshold of 30 using the script Trim.pl
(http://wiki.bioinformatics.ucdavis.edu/index.php/Trim.pl). All reads were screened for adaptor,
barcorde, primer, and transposan sequences and trimmed as needed using FASTX-Toolkit
(http://hannonlab.cshl.edu/fastx_toolkit/index.html). De novo genome assembly was performed
with several assembly programs. Sequences generated through the 454 platform were first
assembled with Roche’s GS De Novo Assembler v 2.6 (“Newbler”) 2 using default parameters.
De novo assemblies of 454 reads were also performed using mira 3 with the default settings for
normal quality de novo genome assembly. De novo assembly of subsets of Illumina reads was
performed with velvet 4, using an estimated coverage of 1000x, kmer size of 21, and a coverage
cutoff of 5). Large contigs from Newbler , mira, and velvet were consolidated using Geneious
Pro v 5.6.6 (Biomatters, Ltd, http://www.geneious.com) and aligned with progressiveMauve 5 to
visualize the relationship of large contigs from different assemblies and to identify gaps to close.
Primers were designed at the ends of contigs using either Geneious Pro or CLC Genomics
Workbench v 5.1 (CLCbio, http://www.clcbio.com) to amplify gaps between contigs. Positive
PCR amplification products linking contigs were cleaned using a Min-Elute PCR Purification kit
(Qiagen) and Sanger sequenced. A nearly complete draft genome from strain JS085 served as a
reference genome for the remaining five strains. Both Illumina and 454 reads were mapped to the
reference genome with CLC Genomics Workbench. Unmapped reads were then assembled de
novo to ensure that novel genomic content in the mapped strains was not overlooked. De novo
assembly of 454 and/or Illumina reads for each strain was also performed in CLC Genomics
Workbench and compared to the mapped assemblies using progressiveMauve.
Four of the strains were sequenced using both 454 and Illumina and two strains were
sequenced only with Illumina. The sequencing coverage depth of quality-filtered reads ranged
from 22X to 50X for 454 and up to 3618X for Illumina (Table 2 in main text). Lebetimonas
strain JS085 had the highest coverage of 454 reads and was assembled into 33 large contigs with
Newbler and 1747 contigs with mira. The 20 largest contigs from each of these assemblies were
consolidated using de novo assembly in Geneious to 10 contigs. An additional round of assembly
in Geneious with the 10 consolidated contigs and velvet contigs greater than 10 Kbp further
consolidated the draft genome to 6 contigs. Primers were designed for all possible combinations
between the 6 contigs. One gap was closed using Sanger-sequenced positive pcr products.
Finally, all 454 and Illumina reads for strain JS085 were mapped to the draft genome consisting
of 5 contigs and the resulting consensus was used as the final draft genome. The five remaining
genomes were assembled by mapping 454 and Illumina reads to the JS085 reference genome in
CLC Genomics Workbench. Hybrid de novo assemblies in CLC Genomics Workbench of each
strain did not extend contigs or close gaps between the 5 contigs of the draft genomes.
Assemblies of unmapped reads produced only short contigs with no significant similarities using
nucleotide BLAST 6.
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Table S1. Proportion of shared genes with >60% identity in Lebetimonas strains.
JH292
JH292
JH369
JS032
JS085
JS138
JS170
96.0%
98.8%
98.8%
99.5%
99.7%
JH369
92.8%
99.0%
99.8%
98.9%
100.7%
JS032
90.2%
93.2%
96.0%
96.2%
96.9%
JS085
90.5%
94.0%
96.4%
96.4%
97.8%
JS138
89.1%
91.5%
94.1%
94.1%
JS170
89.8%
93.3%
95.7%
96.2%
95.6%
94.9%
Table S2. Average nucleotide identity (ANI) of shared genes in Lebetimonas strains.
JH292
JH292
JH369
JS032
JS085
JS138
JS170
95.7%
95.8%
95.8%
96.0%
95.8%
JH369
96.1%
98.5%
99.2%
99.3%
99.3%
JS032
98.8%
98.4%
98.9%
98.3%
98.9%
JS085
96.3%
99.3%
99.0%
99.0%
99.9%
JS138
96.5%
99.2%
98.4%
98.9%
JS170
96.4%
99.3%
99.0%
99.9%
99.0%
98.9%
Figure S1. Photos of representative diffuse flow vents at NW Rota-1 Seamount. A) Arrowhead.
B and C) Marker 109. D) Sulfur Crust.
Figure S2. Dotplot created with nucmer comparing the nucleotide sequences of the two least
similar genomes, Lebetimonas strains JS085 and JH292, showing the synteny of the draft
genomes.