Comparative analysis of four essential Gracilariaceae species in China based on whole transcriptomic sequencing

Three Gracilaria species, G. chouae, G. blodgettii, G. vermiculophylla and a close relative species, Gracilari-opsis lemaneiformis which is now nominated as Gracilaria lemaneiformis, are the typically indigenous spe-cies which are important resources for the production of special proteins, phycobilisomes, special carbo-hydrates, and agar in China. In this study, de novo transcriptome sequencing on these four species using the next generation sequencing technology was performed for the first time. Functional annotations on assembled sequencing reads showed that the transcriptomic profiles were quite different between G. lema-neiformis and other three Gracilaria species. Comparative analysis of differential gene expression related to carbohydrate and phycobiliprotein metabolisms also showed that the expression profiles of these essential genes were different in four species. The genes encoding allophycocyanin, phycocyanin and phycoerythrin were further examined in four species and their deduced amino acid sequences were used for phylogenetic analysis to confirm that G. lemaneiformis had close relationship to genus Gracilaria, as well as that within genus Gracilaria, G. chouae had closer relationship to G. vermiculophylla rather than to G. blodgettii. The de novo transcriptome study on four species provided a valuable genomic resource for further understanding and analysis on biological and evolutionary study among marine algae.

Transcriptome-wide evolutionary analysis on essential brown algae(Phaeophyceae)in China

Brown algae （Chromista, Ochrophyta, Phaeophyceae） are a large group of multicellular algae that play im-portant roles in the ocean＇s ecosystem and biodiversity. However, poor molecular bases for studying their phylogenetic evolutions and novel metabolic characteristics have hampered progress in the field. In this study, we sequenced the de novo transcriptome of 18 major species of brown algae in China, covering six orders and seven families, using the high-throughput sequencing platform Illumina HiSeq 2000. From the transcriptome data of these 18 species and publicly available genome data of Ectocarpus siliculosus and Phaeodactylum tricornutum, we identified 108 nuclear-generated orthologous genes and clarified the phy-logenetic relationships among these brown algae based on a multigene method. These brown algae could be separated into two clades：Clade Ishigeales-Dictyotales and Clade Ectocarpales-Laminariales-Desmares-tiale-Fucales. The former was at the base of the phylogenetic tree, indicating its early divergence, while the latter was divided into two branches, with Order Fucales diverging from Orders Ectocarpales, Laminariales, and Desmarestiale. In our analysis of taxonomy-contentious species, Sargassum fusiforme and Saccharina sculpera were found to be closely related to genera Sargassum and Saccharina, respectively, while Petalonia fascia showed possible relation to genus Scytosiphon. The study provided molecular evidence for the phylo-genetic taxonomy of brown algae.

Phylogenomic analysis of transcriptomic sequences of mitochondria and chloroplasts of essential brown algae(Phaeophyceae)in China

The chloroplast and mitochondrion of brown algae （Class Phaeophyceae of Phylum Ochrophyta） may have originated from different endosymbiosis. In this study, we carried out phylogenomic analysis to distinguish their evolutionary lineages by using algal RNA-seq datasets of the 1 000 Plants （1KP） Project and publicly available complete genomes of mitochondria and chloroplasts of Kingdom Chromista. We have found that there is a split between Class Phaeophyceae of Phylum Ochrophyta and the others （Phylum Cryptophyta and Haptophyta） in Kingdom Chromista, and identified more diversity in chloroplast genes than mitochondrial ones in their phylogenetic trees. Taxonomy resolution for Class Phaeophyceae showed that it was divided into Laminariales-Ectocarpales clade and Fucales clade, and phylogenetic positions of Kjellmaniella crassi-folia, Hizikia fusifrome and Ishige okamurai were confirmed. Our analysis provided the basic phylogenetic relationships of Chromista algae, and demonstrated their potential ability to study endosymbiotic events.

A complete genome assembly of Glaciecola mesophila sp. nov. sequenced by using BIGIS-4 sequencer system

Using a pyrosequencing-based custom-made sequencer BIGIS-4, we sequenced a Gram-negative bacterium Glaciecola mesophila sp. nov. (Gmn) isolated from marine invertebrate specimens. We generated 152043 sequencing reads with a mean high-quality length of 406 bp, and assembled them using the BIGIS-4 post-processing module. No systematic low-quality data was detected beyond expected homopolymer-derived errors. The assembled Gmn genome is 5144318 bp in length and harbors 4303 annotated genes. A large number of metabolic genes correspond to various nutrients from surface marine invertebrates. Its abundant cold-tolerant and cellular signaling and related genes reveal a fundamental adaptation to low-temperature marine environment.

Complete genome sequence of methicillin-sensitive Staphylococcus aureus containing a heterogeneic staphylococcal cassette chromosome element

Staphylococcus aureus is a common human bacterium that sometimes becomes pathogenic,causing serious infections.A key feature of S.aureus is its ability to acquire resistance to antibiotics.The presence of the staphylococcal cassette chromosome(SCC) element in serotypes of S.aureus has been confirmed using multiplex PCR assays.The SCC element is the only vector known to carry the mecA gene,which encodes methicillin resistance in S.aureus infections.Here,we report the genome sequence of a novel methicillin-sensitive S.aureus(MSSA) strain:SCC-like MSSA463.This strain was originally erroneously serotyped as methicillin-resistant S.aureus in a clinical laboratory using multiplex PCR methods.We sequenced the genome of SCC-like MSSA463 using pyrosequencing techniques and compared it with known genome sequences of other S.aureus isolates.An open reading frame(CZ049;AB037671) was identified downstream of attL and attR inverted repeat sequences.Our results suggest that a lateral gene transfer occurred between S.aureus and other organisms,partially changing S.aureus infectivity.We propose that attL and attR inverted repeats in S.aureus serve as frequent insertion sites for exogenous genes.

Improved picoliter-sized micro-reactors for high-throughput biological analysis

High-throughput pyrosequencing,carried out in millions of picoliter-sized reactors on a fiber-optic slide,is known for its longer read length.However,both optical crosstalk(which reduces the signal-to-noise ratio of CCD images)and chemical retention adversely affect the accuracy of chemiluminescence determination,and ultimately decrease the read length and the accuracy of pyrosequencing results.In this study,both titanium and oxidized aluminum films were deposited on the side walls and upper faces of micro-reactor slides to enhance optical isolation;the films reduced the inter-well crosstalk by one order of magnitude.Subsequently,chemical retention was shown to be caused by the lower diffusion coefficient of the side walls of the picolitersized reactors because of surface roughness and random pores.Optically isolated fiber-optic slides over-coated with silicon oxide showed smoother surface morphology,resulting in little chemical retention;this was further confirmed with theoretical calculations.Picoliter-sized micro-reactors coated with titanium-silicon oxide films showed the least inter-well optical crosstalk and chemical retention;these properties are expected to greatly improve the high-throughput pyrosequencing performance.