Draft Genome Sequences of Two Unclassified Chitinophagaceae Bacteria, IBVUCB1 and IBVUCB2, Isolated from Environmental Samples

ABSTRACT We report here the draft genome sequences of two Chitinophagaceae bacteria, IBVUCB1 and IBVUCB2, assembled from metagenomes of surface samples from freshwater lakes. The genomes are >99% complete and may represent new genera within the Chitinophagaceae family, indicating a larger diversity than currently identified.

A dvances in high-throughput sequencing, coupled with decreasing costs, have increased the number of available bacterial genomes almost exponentially. Genome sequencing, however, has traditionally been limited to species that can be held and grown in culture due to the high DNA volumes needed. A predominant focus on cultivable species has led to a genome bias, and, as a result, true bacterial diversity is poorly represented. Metagenomic studies are rectifying this bias and have already revealed a large novel diversity (1). However, metagenomics remains limited, with many ecosystems yet to be sampled. We aim to expand species richness by identifying novel bacteria from varied environmental samples. Here, we present the draft genomes of two unclassified Chitinophagaceae bacteria, which were surface-isolated from freshwater lakes in Norway (Årungen, Ås) and Japan (Tsukuba, Ibaraki).
DNA was isolated using the standard phenol-chloroform protocol with ethanol precipitation and subsequent cleaning using Zymo genomic cleaner and concentrator. DNA was prepared and sequenced on an Illumina HiSeq 2500 (150-bp paired-end reads; 350-bp insert size) and PacBio RS2 with P6-C4 chemistry (20 kb) at the Norwegian Sequencing Centre. Metagenome drafts were assembled using SPAdes version 3.9.0 (2); single genomes were separated with MetaBAT (3); and quality was assessed with CheckM (4). Separate genomes were scaffolded using LINKS (5), and gaps were closed with Sealer (6). Genome assemblies were evaluated with PROmer (7) and REAPER (8) before being improved with Pilon (9). Genomes were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (10). Taxonomical rank was established upon evaluation of CheckM (4), PhyloSift (11), and a megaBLAST search against the NCBInr database.
Chitinophagaceae bacterium IBVUCB1 was assembled into two scaffolds constituting six contigs with a sequence length of 3.41 Mb and a GC content of 42.64%. Scaffold N 50 was 1.92 Mb with an Illumina coverage of 143ϫ and a PacBio coverage of 9ϫ. CheckM estimated genome completeness at 99.01% with no contamination or strain heterogeneity. The genome constitutes 3,056 genes, 43 RNAs, 36 tRNAs, 3 noncoding RNAs (ncRNAs), and 5 pseudogenes.
Chitinophagaceae bacterium IBVUCB2 was assembled into three scaffolds constituting four contigs with a total sequence length of 3.99 Mb and a GC content of 38.42%.
The scaffold N 50 was 2.26 Mb with an Illumina coverage of 32ϫ and a PacBio coverage of 8ϫ. CheckM estimated genome completeness at 99.51% with no contamination or strain heterogeneity. The genome constitutes 3,527 genes, 42 RNAs, 36 tRNAs, 3 ncRNAs, and 25 pseudogenes.
The genomes were confirmed as novel Chitinophagaceae bacteria according to a BLASTn search of 16S queries against the NCBInr database: IBVUCB1 had a 93% identity to Sediminibacterium salmoneum 16S (NR_044197), and IBVUCB2 had a 96% identity to the 16S of the same species. IBVUCB1 and IBVUCB2 had a 94% 16S identity to each other. The low identity to known Chitinophagaceae spp. may suggest IBVUCB1 and IBVUCB2 as new genera, indicating a larger diversity than currently identified.
Accession number(s). The draft genomes of Chitinophagaceae bacteria IBVUCB1 and IBVUCB2 sequenced under this project have been deposited at DDBJ/EMBL/ GenBank under the accession numbers NFUW00000000 and NFUV00000000, respectively. These biosamples (SAMN06840505 and SAMN06840506, respectively) are part of BioProject PRJNA384425.

ACKNOWLEDGMENTS
We thank Abel (UiO), in particular projects nn9244k and nn9404k, for providing computing resources. We also thank the Midas cluster at VIB for providing additional computing resources. We are grateful to the Norwegian Sequencing Centre (NSC) for providing sequencing support and analysis for both Illumina and PacBio. We thank Thomas Haverkamp (IBV, UiO) for discussions related to the assembly and analysis of prokaryote genomes.
This work has been supported by research grants from the Norwegian Research Council to R.J.S.O. (project 230868).