Non-contiguous finished genome sequence of Phocaeicola abscessus type strain 7401987T

Phocaeicola abscessus strain 7401987T is the sole member of the genus Phocaeicola. This bacterium is Gram-negative, non-spore-forming, coccoid to rod-shaped and motile by lophotrichous flagella. It was isolated from a human brain abscess sample. In this work, we describe a set of features of this organism, together with the complete genome sequence and annotation. The 2,530,616 bp long genome contains 2,090 protein-coding genes and 54 RNA genes, including 4 rRNA operons.


Introduction
Phocaeicola abscessus strain 7401987 T (CSUR P22 T = DSM 21584 T = CCUG 55929 T ) is the type strain of P. abscessus. This bacterium was isolated from a brain abscess sample from a 76-year-old patient who underwent neurosurgical intervention after cancer of the face [1]. It is a Gramnegative strictly anaerobic coccoid to rod-shaped bacterium. Currently, the genus Phocaeicola contains only one species [2]. Here we present a summary classification and a set of features for P. abscessus, together with the description of the non-contiguous finished genomic sequencing and annotation.

Genome project history
The organism was selected for sequencing on the basis of its phylogenetic position and 16S rRNA similarity to other members of the order Bacteroidales and is part of study of the new species characterized in our laboratory. A summary of the project information is shown in Table 2. The EMBL accession number is CAKQ01000000 and consists of 39 contigs (≥ 500 bp) and 9 scaffolds. Table 2 shows the project information and its association with MIGS version 2.0 compliance. , not directly observed for the living, isolated sample, but based on a g enerally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [9]. If the evidence is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledg ements.

Genome sequencing and assembly
Shotgun and 3-kb paired-end sequencing strategies were performed. The shotgun library was constructed with 500 ng of DNA with a GS Rapid library Prep kit (Roche). For the paired-end sequencing, 5 µg of DNA was mechanically fragmented on a Hydroshear device (Digilab) with an enrichment size at 3-4 kb. The DNA fragmentation was visualized using a 2100 BioAnalyzer (Agilent) on a DNA labchip 7500 with an optimal size of 3.1 kb. The library was constructed according to the 454 GS FLX Titanium paired-end protocol. Circularization and nebulization were performed and generated a pattern with an optimal size of 579 bp. After PCR amplification through 17 cycles followed by double size selection, the single stranded paired-end library was then quantified using a Genios fluorometer (Tecan) at 8,770 pg/µL. The library concentration equivalence was calculated as 1.39E+10 molecules/µL. The library was stored at -20°C until further use. The shotgun and paired-end libraries were clonally-amplified with 0.5 cpb and 2 cpb in 3 and 2 SV-emPCR reactions with the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche). The yields of the emPCR were 9.63% and 10.3%, respectively, in the 5 to 20% range from the Roche procedure. Approximately 790,000 beads for the shotgun application and for the 3kb paired end were loaded on a GS Titanium PicoTiterPlate PTP Kit 70x75 and sequenced with a GS FLX Titanium Sequencing Kit XLR70 (Roche). The run was performed overnight and then analyzed on the cluster through the gsRunBrowser and Newbler assembler (Roche). A total of 311,276 passed filter wells were obtained and generated 35.9 Mb with a length average of 282 bp. The passed filter sequences were assembled using Newbler with 90% identity and 40 bp as overlap. The final assembly identified 9 scaffolds and 39 contigs (>500 bp).

Genome annotation
Open Reading Frames (ORFs) were predicted using Prodigal [10] with default parameters but the predicted ORFs were excluded if they were spanning a sequencing GAP region. The predicted bacterial protein sequences were searched against the GenBank database [11] and the Clusters of Orthologous Groups (COG) databases [12] using BLASTP. The tRNAscan-SE tool [13] was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer [14]. Transmembrane domains and signal peptides were predicted using TMHMM [15] and SignalP [16], respectively. ORFans of alignment length greater than 80 amino acids were identified if their BLASTp E-value was lower than 1e-03. If alignment lengths were smaller than 80 amino acids, we used an E-value of 1e-05. Such parameter thresholds have been used in previous works to define ORFans.
To estimate the mean level of nucleotide sequence similarity at the genome level between P. abscessus and Prevotella timonensis, Bacteroides thetaiotaomicron and Paraprevotella clara, we compared the ORFs using only comparison sequences in the RAST server [17] at a query coverage of ≥70% and a minimum nucleotide length of 100 bp.

Genome properties
The genome is 2,530,616 bp long with a 47.31% GC content (Table 3, Figure 3). Of the 2,144 predicted genes, 2,090 were protein-coding genes, and 54 were RNAs. A total of 1,464 genes (70.05%) were assigned a putative function. A total of 112 genes were identified as ORFans (5.39%). The remaining genes were annotated as hypothetical proteins (436 genes (20.86%)). The remaining genes were annotated as either hypothetical proteins or proteins of unknown functions. The distribution of genes into COGs functional categories is presented in Table 4. The properties and the statistics of the genome are summarized in Tables 3 and 4. Two CRISPRs were found using CRISPERfinder program online [18]. The first one on contig 1 includes at least 3 predicted spacer regions and the second one on contig 18 includes at least 53 predicted spacer regions. a) The total is based on either the size of the g enome in base pairs or the total number of protein coding g enes in the annotated genome.

Figure 3.
Graphical circular map of Phocaeic ola abscessus g enome. From outside to the center: Genes on the forward strand colored by COG categ ories (only g enes assig ned to COG), g enes on the reverse strand col ored by COG categ ories (only g ene assig ned to COG), RNA genes (tRNAs g reen, rRNAs red), GC content and GC skew (three circles), GC content. The total is based on the total number of protein coding g enes in the annotated genome.

Comparison with other genomes
Phocaeicola abscessus is the sole bacterium included in the genus Phocaeicola. We compared the genome of P. abscessus with those of Prevotella timonensis (CBQQ010000001) Paraprevotella clara (AFFY01000000) and Bacteroides thetaiotaomicron (AE015928.1). P. abscessus showed a mean nucleotide sequence similarity of 76.40%, 77.06% and 77.52% at the genome level (range 70-92.25%, 70.04-95.51% and 70.04-93.02%) with P. timonensis, P. clara and B. thetaiotaomicron, respectively. Presently, the family to which P. abscessus belongs is undetermined and the sole comparison based on nucleotide sequence similarity may not be sufficient to answer this question. In the future, further comparison of the genomes will allow us to find traits to classify the genus Phocaeicola in one of these 3 families or to create a new family, the family Phocaeicolaceae.