Nextera mate pair

R. marinus strain MAT378/R-21 is a close relative of the type strain R. marinus DSM4252. Both strains were originally isolated from a shallow submarine hot spring in SW Iceland (Alfredsson et al., 1988 (link)). The MAT378 strain was cultivated in RSC selective medium (Bjornsdottir et al., 2007 (link)), containing 0.2% soluble starch (Merck). Pure high-molecular-weight DNA and plasmid DNA was isolated using the MasterPure Gram Positive DNA Purification kit (Lucigen) according to manufacturer’s instructions and used for genome sequencing. The genome was originally sequenced by Sanger sequencing with approximately 5x coverage. With the emergence of high throughput sequencing, the genome was re-sequenced. Libraries were prepared using the Nextera Flex and Nextera MatePair methods (Illumina) and sequenced on the Illumina MiSeq platform. The generated sequence reads were assembled by using SPAdes assembler (Bankevich et al., 2012 (link)) and annotated by using the RAST annotation server (Overbeek et al., 2014 (link)).

The isolates were subjected to whole-genome sequencing using both paired-end libraries and mate-pair libraries, in order to create high-quality genomes, especially with respect to the mobilome. The isolates were run on Illumina Miseq 2000 2 × 250 bp (500 cycles) after library preparation with Nextera XT (paired-end libraries) and Nextera Mate Pair libraries (Illumina), respectively. The genomes were assembled with Allpaths-LG with the following settings: scaffolding, insert size paired-end 300 ± 200 and 3,000 ± 1,000 for mate pair. The sequencing and following assembly yielded high-quality genomes with low scaffold counts (Supplementary Table 1). Genomes were annotated with Prokka v 1.12.

The DNA was isolated and purified using the EZ1 DNA Tissue Kit (BioRobot EZ1 Advanced XL instrument, Qiagen, Hilden, Germany) following the manufacturer’s instructions. Genomic DNA of the four Lebanese isolates was sequenced using the MiSeq Technology (Illumina Inc, San Diego, CA, USA). Briefly, the genomic DNA was quantified by the Qubit assay with the high sensitivity kit (Life technologies, Carlsbad, CA, USA) and 0.2 µg/µL of the DNA was used for sequencing. The DNA was fragmented and amplified by a limited PCR (12 cycles), introducing dual-index barcodes and sequencing adapters. After purification on AMPure XP beads (Beckman Coulter Inc, Fullerton, CA, USA), the libraries were normalized and pooled for sequencing on the Illumina MiSeq platform (Illumina Inc., San Diego, USA). Paired-end sequencing and automated cluster generation with dual indexed 2 × 250-bp reads were performed for 40 h run. Total information of 8.2 Gb was obtained from a 1,207,000/mm² cluster density with a cluster passing quality control filters of 89.3% (10507.2 passed filtered reads). The mate pair library was prepared with 1.5 µg of genomic DNA using the Nextera Mate-Pair Illumina guide. The genomic DNA sample was simultaneously fragmented and tagged with a mate-pair junction adapter.