Abstract
We present the draft genome sequences for 26 strains of Porphyromonas (P. canoris, P. gulae, P. cangingavalis, P. macacae, and 7 unidentified) and an unidentified member of the Porphyromonadaceae family. All of these strains were isolated from the canine oral cavity, from dogs with and without early periodontal disease.
GENOME ANNOUNCEMENT
Members of the Porphyromonas genus have been shown to be present in periodontal disease in both humans and dogs (1, 2). As part of a larger study on canine periodontal disease (1), a number of Porphyromonas strains were isolated, cultured, and sequenced.
The study cohort comprised client-owned pet dogs that presented at a veterinary referral dental clinic (The Veterinary Dental Surgery, Surrey, United Kingdom). Only dogs under anesthetic for routine dental treatment or other noninfectious conditions were included in the study. No dogs were anesthetized solely for the collection of plaque samples. For further details see our previously published work on this cohort (1).
Bacterial isolates were grown on Columbia blood agar (CBA) containing 5% defibrinated horse blood supplemented with 5 mg/L Hemin (catalog no. H9039; Sigma) and 0.5 mg/L Menadione (catalog no. M5625; Sigma). The isolates were incubated at 38°C in an anaerobic cabinet (DonWhitley Scientific Ltd., Shipley, United Kingdom) (80% nitrogen, 10% hydrogen, and 10% carbon dioxide) for 1 to 21 days. DNA extraction was performed on scrapings resuspended in 3 mL brain heart infusion broth (BHI) (catalog no. CM1135; Oxoid), using the Joint Genome Institute DNA isolation bacterial cetyltrimethylammonium bromide (CTAB) protocol.
Following genomic DNA extraction, 16S rRNA genes were amplified by PCR using 16S universal primers. Two forward primers, AC84 (5′ AGA GTT TGA TYM TGG CTC AG 3′) and AC83 (5′ AGG GTT CGA TTC TGG CTC AG 3′, which contains sequence specific to the Bifidobacteriaceae), were used. Both primers are homologous to Escherichia coli position 8 to 27. The reverse primer was C72 (5′ GYT ACC TTG TTA CGA CTT 3′), which is homologous to E. coli position 1492 to 1509.
Two Illumina sequencing library preparation protocols were used, one based on mechanical shearing of DNA, and another based on tagmentation. The tagmentation libraries were constructed using the Nextera DNA sample prep kit (Epicentre) according to the manufacturer’s instructions. The libraries were size selected (300 to 600 nucleotides) on a PippinPrep instrument (Sage Science). For the mechanical shearing libraries, genomic DNA was subjected to sonication using a Bioruptor sonication device (Diagenode) programmed to generate 200 to 300 nucleotide fragments. These fragments were then transferred to an automated DNA library preparation platform Apollo 324 (IntegenX), where steps of end-repair, A-tailing, and bar code adapter ligation were carried out. Subsequently, adapter-ligated samples were subjected individually to 11 cycles of PCR amplification (with a Qiagen kit, [catalog no. 201205]), cleaned up, and size selected (320 bp) on a PippinPrep device (Sage Bioscience). All libraries were sequenced on an Illumina HiSeq 2000 machine.
All sequence processing and assembly of the Illumina reads were performed using the A5 assembly pipeline (3). Automated annotation was performed using the RAST annotation server (4). The assembly and annotation statistics are presented in Table 1.
Table 1.
Strain identifier | Accession no. | No. of contigs | No. of scaffolds | Genome size (bp) | N50 | No. of raw reads | Coverage (×) |
---|---|---|---|---|---|---|---|
Porphyromonas canoris COT-108_OH1224 | JQZX00000000 | 93 | 21 | 2,308,674 | 356,707 | 11,771,344 | 358 |
Porphyromonas sp. COT-108_OH1349 | JRAH00000000 | 126 | 43 | 2,330,607 | 294,161 | 6,895,102 | 194 |
Porphyromonas gulae I COT-052_OH1355 | JRAG00000000 | 151 | 40 | 2,340,299 | 162,065 | 9,130,698 | 357 |
Porphyromonas cangingivalis COT-109_OH1379a | JQJF00000000 | 58 | 21 | 2,364,758 | 227,673 | 6,682,444 | 205 |
Porphyromonas cangingivalis COT-109_OH1386 | JQJD00000000 | 145 | 66 | 2,441,844 | 100,405 | 6,061,670 | 149 |
Porphyromonas gingivicanis COT-022_OH1391a | JQZW00000000 | 53 | 20 | 1,983,669 | 255,048 | 8,862,612 | 329 |
Porphyromonas sp. COT-239_OH1446 | JRAO00000000 | 62 | 37 | 1,961,776 | 133,546 | 4,575,892 | 150 |
Porphyromonas crevioricanis COT-253_OH1447 | JQJC00000000 | 63 | 30 | 2,161,087 | 144,469 | 8,455,142 | 263 |
Porphyromonas gulae I COT-052_OH1451 | JRAI00000000 | 134 | 91 | 2,463,757 | 46,861 | 13,491,026 | 371 |
Porphyromonas crevioricanis COT-253_OH2125 | JQJB00000000 | 76 | 14 | 2,105,287 | 491,012 | 8,101,504 | 358 |
Porphyromonas gulae I COT-052_OH2179 | JRAJ00000000 | 165 | 26 | 2,442,276 | 247,543 | 13,797,658 | 527 |
Porphyromonas macacae COT-192_OH2631 | JRFB00000000 | 102 | 54 | 2,316,420 | 1,255,122 | 14,360,788 | 572 |
Porphyromonas canoris OH2762 | JQZV00000000 | 80 | 14 | 2,202,536 | 351,685 | 12,612,176 | 539 |
Porphyromonas gulae II COT-052_OH2857 | JRFD00000000 | 143 | 53 | 2,333,470 | 78,104 | 15,364,628 | 597 |
Porphyromonas macacae COT-192_OH2859 | JRFA00000000 | 129 | 34 | 2,364,070 | 207,096 | 11,085,904 | 426 |
Porphyromonas sp. COT-108_OH2963 | JRAP00000000 | 53 | 21 | 2,179,370 | 225,693 | 5,782,088 | 164 |
Porphyromonas gulae OH3161B | JQJE00000000 | 161 | 47 | 2,335,601 | 149,377 | 12,527,276 | 500 |
Porphyromonas gulae II COT-052_OH3439 | JRAK00000000 | 290 | 163 | 2,588,710 | 55,832 | 7,989,956 | 192 |
Porphyromonas gulae I COT-052_OH3471 | JRAQ00000000 | 129 | 44 | 2,371,923 | 134,999 | 4,743,702 | 123 |
Porphyromonas gulae II COT-052_OH3498 | JRAF00000000 | 102 | 71 | 2,252,877 | 67,961 | 8,041,104 | 312 |
Porphyromonas sp. COT-290_OH3588CRE | JRFC00000000 | 105 | 48 | 2,294,016 | 183,859 | 14,279,656 | 588 |
Porphyromonas gulae II COT-052_OH3856 | JRAT00000000 | 117 | 31 | 2,388,773 | 131,574 | 14,306,028 | 557 |
Porphyromonas gulae II COT-052_OH4119 | JRAL00000000 | 143 | 52 | 2,287,427 | 154,457 | 12,704,688 | 518 |
Porphyromonadaceae [G-1] sp. COT-184_OH4590 | JRAN00000000 | 191 | 79 | 2,392,483 | 66,072 | 6,627,810 | 178 |
Porphyromonas sp. UQD_349_COT-052_OH4946 | JQZY00000000 | 131 | 34 | 2,384,876 | 169,169 | 10,619,640 | 414 |
Porphyromonas sp. COT-290_OH860 | JRAR00000000 | 122 | 82 | 2,343,073 | 60,215 | 7,717,532 | 206 |
These libraries were constructed with mechanical shearing; all others were constructed by tagmentation.
Nucleotide sequence accession numbers.
All 26 assemblies described in this paper have been deposited as whole-genome shotgun projects in DDBJ/EMBL/GenBank under the accession numbers provided in Table 1.
ACKNOWLEDGMENTS
Bacterial strains and genomic DNA were isolated and the species confirmed at the Waltham Centre for Pet Nutrition. Illumina sequencing was performed at the DNA Technologies Core facility in the Genome Center at University of California, Davis, CA.
Funding for this work was provided by Waltham Centre for Pet Nutrition.
We thank John Zhang and Dave Larsen for help with library preparation.
Footnotes
Citation Coil DA, Alexiev A, Wallis C, O’Flynn C, Deusch O, Davis I, Horsfall A, Kirkwood N, Jospin G, Eisen JA, Harris S, Darling AE. 2015. Draft genome sequences of 26 Porphyromonas strains isolated from the canine oral microbiome. Genome Announc 3(2):e00187-15. doi:10.1128/genomeA.00187-15.
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