Chromosomal-level assembly of the Asian Seabass genome using long sequence reads and multi-layered scaffolding

We report here the ~670 Mb genome assembly of the Asian seabass (Lates calcarifer), a tropical marine teleost. We used long-read sequencing augmented by transcriptomics, optical and genetic mapping along with shared synteny from closely related fish species to derive a chromosome-level assembly with a contig N50 size over 1 Mb and scaffold N50 size over 25 Mb that span ~90% of the genome. The population structure of L. calcarifer species complex was analyzed by re-sequencing 61 individuals representing various regions across the species' native range. SNP analyses identified high levels of genetic diversity and confirmed earlier indications of a population stratification comprising three clades with signs of admixture apparent in the South-East Asian population. The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics

CLONING AND CHARACTERIZATION OF GENES ENCODING CARDIOTOXINS IN NAJA NAJA SPUTATRIX

Ph.DDOCTOR OF PHILOSOPH

Long-read whole genome sequencing and comparative analysis of six strains of the human pathogen Orientia tsutsugamushi.

BackgroundOrientia tsutsugamushi is a clinically important but neglected obligate intracellular bacterial pathogen of the Rickettsiaceae family that causes the potentially life-threatening human disease scrub typhus. In contrast to the genome reduction seen in many obligate intracellular bacteria, early genetic studies of Orientia have revealed one of the most repetitive bacterial genomes sequenced to date. The dramatic expansion of mobile elements has hampered efforts to generate complete genome sequences using short read sequencing methodologies, and consequently there have been few studies of the comparative genomics of this neglected species.ResultsWe report new high-quality genomes of O. tsutsugamushi, generated using PacBio single molecule long read sequencing, for six strains: Karp, Kato, Gilliam, TA686, UT76 and UT176. In comparative genomics analyses of these strains together with existing reference genomes from Ikeda and Boryong strains, we identify a relatively small core genome of 657 genes, grouped into core gene islands and separated by repeat regions, and use the core genes to infer the first whole-genome phylogeny of Orientia.ConclusionsComplete assemblies of multiple Orientia genomes verify initial suggestions that these are remarkable organisms. They have larger genomes compared with most other Rickettsiaceae, with widespread amplification of repeat elements and massive chromosomal rearrangements between strains. At the gene level, Orientia has a relatively small set of universally conserved genes, similar to other obligate intracellular bacteria, and the relative expansion in genome size can be accounted for by gene duplication and repeat amplification. Our study demonstrates the utility of long read sequencing to investigate complex bacterial genomes and characterise genomic variation

Long-read whole genome sequencing and comparative analysis of six strains of the human pathogen <i>Orientia tsutsugamushi</i>

<div><p>Background</p><p><i>Orientia tsutsugamushi</i> is a clinically important but neglected obligate intracellular bacterial pathogen of the Rickettsiaceae family that causes the potentially life-threatening human disease scrub typhus. In contrast to the genome reduction seen in many obligate intracellular bacteria, early genetic studies of <i>Orientia</i> have revealed one of the most repetitive bacterial genomes sequenced to date. The dramatic expansion of mobile elements has hampered efforts to generate complete genome sequences using short read sequencing methodologies, and consequently there have been few studies of the comparative genomics of this neglected species.</p><p>Results</p><p>We report new high-quality genomes of <i>O</i>. <i>tsutsugamushi</i>, generated using PacBio single molecule long read sequencing, for six strains: Karp, Kato, Gilliam, TA686, UT76 and UT176. In comparative genomics analyses of these strains together with existing reference genomes from Ikeda and Boryong strains, we identify a relatively small core genome of 657 genes, grouped into core gene islands and separated by repeat regions, and use the core genes to infer the first whole-genome phylogeny of <i>Orientia</i>.</p><p>Conclusions</p><p>Complete assemblies of multiple <i>Orientia</i> genomes verify initial suggestions that these are remarkable organisms. They have larger genomes compared with most other Rickettsiaceae, with widespread amplification of repeat elements and massive chromosomal rearrangements between strains. At the gene level, <i>Orientia</i> has a relatively small set of universally conserved genes, similar to other obligate intracellular bacteria, and the relative expansion in genome size can be accounted for by gene duplication and repeat amplification. Our study demonstrates the utility of long read sequencing to investigate complex bacterial genomes and characterise genomic variation.</p></div

The number of core gene groups and the total number of gene groups (including the core gene groups) as more strains are added to the analysis.

<p>Boxplots represent all possible combinations of the number of strains given on the x-axis.</p

Each arrow represents the location of a core gene island containing one or more core genes which are conserved in the same order within an island across all strains.

<p>The arrows are coloured relative to their order in the Karp genome.</p

Predicted gene numbers in different strains of <i>O</i>. <i>tsutsugamushi</i>.

<p>Predicted gene numbers in different strains of <i>O</i>. <i>tsutsugamushi</i>.</p

Annotation statistics of the Asian seabass genome.

<p>Annotation statistics of the Asian seabass genome.</p

Scaffolding using optical map, genetic map and synteny with closely related fish genomes produced chromosome-level assembly of the Asian seabass genome.

<p>(A) Comparison of <i>L</i>. <i>calcarifer</i> to two closely related fish species (<i>G</i>. <i>aculeatus</i>, and <i>D</i>. <i>labrax</i>) at the genome-wide level. Colours used for depicting assembled chromosomes are random for each of the three genomes. Different colours in a single <i>L</i>. <i>calcarifer</i> linkage group are used to represent the inter-chromosomal rearrangements. Black arcs show collinear blocks that are intra-chromosomally rearranged between the species. (B) Genome assembly (middle panel) shown anchored to two (LG15 and LG18) of the twenty four <i>L</i>. <i>calcarifer</i> linkage groups while the right panel represents the scaffolded assembly (regions in grey depict the additional contigs brought together by scaffolding).</p