(The GenBank accession number for the 16S rRNA sequence of L2TR^T is AF288370)

The microorganism was the first new bacterial species discovered in Hawaii. It was collected in Pele's Pit, 4,376 feet below the ocean's surface on the summit of underwater volcano Loihi, off the Big Island with the submersible Pisces V in 1999.

They were able to cultivate some of the bacteria for the first time, and a genetic analysis showed they had discovered an unknown species. It was named Idiomarina loihiensis (called L2-TR in the lab).

The I. loihiensis genome sequence was done using a hybrid of whole-genome random shotgun library and BAC clone library approaches. Genomic DNA of I. loihiensis was isolated and randomly sheared by controlling the sonication time, followed by size-fractionation on the agarose gel. DNA fragments at appropriate sizes from the gel were excised and extracted from the agarose gel. The size selected DNA fragments were ligated into subvectors using Ready-To -Go TM pUC118 vector. 1Kb, 2Kb, and 5Kb whole genome random shotgun libraries were constructed. For BAC clone library, 10 BAC clones were subsequently used for 1 Kb shotgun libraries.

Approximately total of 83,000 clones from the whole genome shotgun libraries and BAC clone libraries were sequenced from both ends by dye-terminator using CEQ2000 automated sequencers (Beckman Coulter).

A total of 69,020 valid sequences were assembled into 148 contigs using the Paracel Genome Assembler (PGA) (Paracel, Pasadena, CA) for the initial assembly, giving about 10-fold coverage of the genome. This assembly resulted in 14 scaffolds that are linked by gap-spanning forward and reverse clone pairs. These scaffolds were further linked to each other using BAC end pairs.

AUTOFINISH module of CONSED was then used to automatically pick primers to close gaps between the contigs by primer-walking. A total of 6 rounds of AUTOFINISH experiments were done and 858 primer-walking sequences were obtained. 858 new sequences were assembled with the previously obtained contigs by PGA through directed assembly using Seqman II from DNAStar version 5.05 (DNAStar, Madison, WI).

This step enabled closure of 126 gaps between the scaffolds and resulting 22 large contigs. For the final gap closing, the direct sequencing of genomic DNA was performed by Fidelity Inc. according to Slesarev et al. A total of 102 direct genomic sequences were obtained and assembled, allowing to close 16 gaps and left 6 large contigs. Assembly of further 131 sequences from sequencing of PCR products that bridge the remaining 6 gaps resulted in a single contig with 2,839,318 base pairs in size.

Low quality regions within the contigs were inspected using CONSED program and such regions were re-sequenced for high quality and more coverage. Finally, the whole genome assembly was verified by 187 long PCR amplifications (Takara Bio Inc, Japan) of the 15-20 kb fragments throughout the genome with 1kb overlapping.